The distribution of lipids between the two sides of biological membranes has been extensively studied and there is now much evidence demonstrating that phospholipids are distributed asymmetrically between the two halves of the membrane bilayer in both bacterial and mammalian plasma membranes (Bretscher, 1972; Rothman & Kennedy, 1977). The distribution of cholesterol in membranes is more controversial. Some workers have claimed an asymmetric distribution of cholesterol in erythrocyte membranes (Fisher, 1971) and also in myelin by using X-ray-diffraction techniques (Caspar 8t Kirschner, 197 1). On the other hand, Blau & Bittman (1978) maintain that cholesterol distribution in erythrocyte membranes is symmetrical. We have measured the distribution of cholesterol between the two sides of rat sciatic-nerve myelin membranes by feeding rats on diets containing deuterium-labelled cholesterol and recording neutron-diffraction patterns from the sciatic nerves of the rats. The centrosymmetric arrangement of myelin with its alternating double layers of the cytoplasmic and of the extracellular sides of the bilayer leaflet permits the distribution of cholesterol between the two halves of the bilayer to be determined unambiguously if sufficient quantities of deuterated cholesterol can be introduced into the nerve. The neutronscattering amplitude of deuterium is greatly different from that of hydrogen and so deuterium in cholesterol provided a label analogous to the heavy-atom label used in X-ray crystallography (Worcester, 1976). Cholesterol labelled with five deuterium atoms at the 2-, 3and 4-positions (adjacent to the hydroxy group) was prepared by enolization exchange on cholest-4-en-3-one in a hot alkaline mixture of singly deuterated ethanol and 2H,0 followed by the reduction of the enol acetate derivative with sodium borodeuteride. The isotopic enrichment of the cholesterol at the 2-, 3and 4-positions was greater than 90%. The crude product was recrystallized from acidified ethanol and the final product was 92% cholesterol (cholest-5-en-3/l-ol), the remainder being epicholesterol (cholest-5-en-3 a-01). Deuterated cholesterol had to be available to the myelin during development so that sufficient quantities were introduced into the membranes for the present study. Female rats were fed beginning 2-3 days before pregnancy on diets containing l o g of deuterated cholesterol/kg of diet, or normal cholesterol for control. Small amounts of [7(n)-3H1cholesterol were added so that specific incorporation of the dietary cholesterol into the sciatic nerves could be determined and was found to be lo%, although incorporation into the central nervous system was less than 1%. The other non-deuterated 90% arose as the result of endogenous synthesis of cholesterol. Feeding was continued throughout pregnancy and after birth up to 3 weeks after weaning. The young rats (about 12 labelled and 12 control) were then slaughtered and sciatic nerves were dissected into KrebsRinger original phosphate buffer (pH 7.4). Neutron-diffraction measurements were made at the Institut Laue-Langevin, Grenoble, France, by using the small-angle scattering instrument, model D 17. Several nerves were mounted together in special cells with thin glass windows and formed samples of dimensions 1.3 cm x 0.6 cm and 0.1 cm thick. Throughout the experiments, the nerves were maintained in circulating Krebs-Ringer phosphate buffer at 15 OC. Neutrons of wavelength 1 nm with 5% wavelength spread were diffracted by the samples and detected on a two-dimensional position-sensitive detector. The detector could be set to measure different parts of the diffraction pattern successively, usually Bragg orders 1-4 in one setting and then 4-7 in another. We recorded seven orders of diffraction from the 18nm repeat unit of myelin in different mixtures of H,O/*H,O Krebs-Ringer buffers. Measuring times were 3 h or less. Structure factors were calculated from the integrated intensities after correction for absorption and with a Lorentz factor inversely proportional to the Bragg order. The structure factors for each order are linear with H,0/2H,0 mixtures except for the fourth order, which shows pronounced multiple scattering effects in high ,H,O mixtures. The linear change of the structure factors provided a means of direct scaling of the labelled nerve and the controls. We were thereby able to calculate the Fourier profile of the labelled and unlabelled membranes on the same scale and by using previously determined phases. The differences between the profile of the labelled membranes and that of the controls shows that the cholesterol is symmetrically distributed between the two halves of the membrane bilayer. The profile of the control membrane shows a nearly symmetrical lipid bilayer so that almost equal amounts of phospholipid are present on each side of the bilayer leaflet as was previously shown by X-ray diffraction (Caspar & Kirschner, 1971; Nelander & Blaurock, 1978; Blaurock & Nelander, 1979). If there is an asymmetry of phospholipids in myelin as in other membranes, then this does not influence the distribution of cholesterol.