The water spaces in cholinergic synaptic vesicles isolated from the electromotor terminals of Torpedo marmorata electric organ have been determined as a fraction of the total vesicle volume by measuring the density changes induced in the vesicles by the addition of permeating substances to iso-osmotic density gradients. Three permeating substances were selected for study: deuterium oxide, dimethylsulphoxide and glycerol. The water spaces measured by these three substances were not equal, being 83%, 72% and 65% of the vesicle volume, respectively. When vesicles were lysed in dilute (10 mM) Hepes buffer (pH 7.0), the deuterium oxide space was not detectably changed, but the dimethylsulphoxide and glycerol spaces assumed the same value of 74–75%. This was interpreted to mean that lysis resulted in the loss of highly hydrated core constituents (presumably mainly acetylcholine and adenosine 5'-triphosphate) whose bound water can be replaced by dimethylsulphoxide and deuterium oxide but not by glycerol. When intact or lysed vesicles were exposed to highly hyperosmotic CsCl gradients, the changes in the density and in the deuterium oxide and glycerol spaces showed that the vesicles had undergone collapse due to osmotic dehydration; 91–96% of the glycerol space is osmotically active water. The density of the membrane was estimated to be 1.11 to 1.135 depending on its protein content. These results confirm, by an independent method, conclusions already reached in this laboratory from the protein and lipid analysis of vesicles ( Ohsawa, Dowe, Morris & Whittaker, Brain Res. 161, 447–457, 1979) and from density measurements at varying osmotic pressures ( Breer, Morris & Whit-taker, Eur. J. Biochem. 87, 453–458, 1978), namely, that the vesicle is a highly hydrated structure with a diameter of 80–100 nm and a lipid-rich membrane 4–5 nm in thickness. The implications of the results for measurements of vesicular membrane potential and intravesicular pH are discussed.