In addition to the well-characterized changes in enzyme activity (Pennell, 1964) and lipid content (Prankerd, 1958), which accompany aging of erythrocytes in vivo, there is some evidence to suggest that changes in the oligosaccharide chains of the cell surface also occur. Aged cells showed a decreased electrophoretic mobility when compared with a young fraction of the erythrocyte population (Danon et al., 1971). The decreased surface charge was related to a decreased density of staining of the cell membrane of old cells with a positively charged colloidal iron suspension. Since the surface charge of erythrocytes is largely attributable to sialic acid (Eylar et al., 1962) these results suggest that aging of erythrocytes is accompanied by loss of sialic acid. Danon et al. (1971) have proposed that removal of sialic acid is a major factor in the elimination of old erythrocytes from the circulation. Jancik & Schauer (1974) have demonstrated that removal of 60% of the sialic acid from rabbit erythrocytes results in their rapid removal from the circulation. In view of the observations suggesting a possible relationship between the life span of erythrocytes and cell-surface carbohydrate, we have measured the sialic acid content of human erythrocytes of different ages isolated from a single individual and have examined the cells for changes in their surface carbohydrate by examination of their interaction with lectins. Erythrocytes can be fractionated into populations with different specificgravity by discontinuous-gradient ultracentrifugation in iso-osmotic solutions of bovine plasma albumin (Piomelli et al., 1967). It has been demonstrated by labelling studies that the specific gravity of erythrocytes increases progressively with age. Washed erythrocytes from freshly drawn human blood were fractionated on discontinuous density gradients of albumin by a modification of the method of Piomelli et al. (1967). Table 1 shows a typical distribution of cells. That fractions contained cells of different average age was confirmed by assaying the activity of glucose 6-phosphate dehydrogenase (Piomelli et a/., 1968). Activity was determined as the rate of change of absorbance at 340nm per mg of haemoglobin. Haemoglobin was measured by the method of Van Kampen & Zijlstra (1961). The haemoglobin content per cell was constant for cells of different age. N-Acetylneuraminic acid was determined by the method of Aminoff (1961) after release from erythrocytes either by Clostridium perfringens neuraminidase or by acid hydrolysis. The conditions used for acid hydrolysis were standardized to allow for the effect of haemoglobin on the pH of the reaction mixture and protein was precipitated before analysis by addition of 5 % (w/v) phosphotungstic acid. Analysis of sialic acid released from replicate samples of erythrocytes from a single individual gave a value of 348+5 (s.D., for eight determinations) ng of N-acetylneuraminic acid per mg of haemoglobin. The oldest and most dense cell fractions consistently showed a sialic acid content which was lower by approx. 15 % than that of the cells at the top of the gradient. This loss of sialic acid is substantially greater than the total lipid-bound sialic acid of erythrocytes, which amounts to no more than 2.5 % of the total erythrocytesialicacid. Thus, although old cells are known to be deficient in lipid (Prankerd, 1958), this alone cannot account for the diminished content of sialic acid. The conclusion that sialic acid is lost from erythrocyte glycoprotein is supported by the observation of Balduini et al. (1974) that glycopeptides isolated from membranes of aged erythrocytes showed significant decreases in sialic acid when compared with glycopeptides from young cells. The agglutination of erythrocytes of blood group 0 of different age has been examined using wheat-germ agglutinin and a lectin partially purified from the haemolymph of Carcinus. A progressive increase in agglutination with cell age was observed with both lectins. Wheat-germ agglutinin has a specificity directed towards carbohydrate groups