The exchange of lecithin between rabbit erythrocytes and serum has been investigated. The erythrocyte lecithin was labelled in vivo by administration of Na332PO4, which resulted in the acquisition of [32PJlecithin by the cells primarily by exchange, and in vitro by esterification with [3H]palmitate, [14C]linoleate, or [14C]linolenate. The rate of exchange of erythrocyte [32PJ-phospholipids was independent of specific serum lipoprotein fractions, but was related to the serum phospholipid concentration. Analysis of the erythrocyte inward and outward exchange rates of [32P]lecithin revealed that only 55% of the cellular lecithin pool takes part in the exchange. A similar conclusion can be reached by comparison of the fractional exchange rates of the acyl- and 32P-labelled erythrocyte lecithin, or by comparison of the ratio of acyl to 32P radioactivity of the erythrocyte lecithin with the ratio found in the serum after exchange. These data indicated that the proportion of the acyl-labelled erythrocyte lecithin pool (± S.E.M.) capable of exchange was 53 ± 5%, 64 ± 12%, and 95 ± 6%, when esterified with [14C]linoleate, [3H]palmitate, and [14C]linolenate, respectively. The differences in the proportion of the acyl-labelled erythrocyte lecithin pool undergoing exchange could not be attributed to the preferential exchange of a particular molecular species of lecithin, although the tetraenoic lecithin did exchange to a relatively greater degree. This suggests that cellular lecithin formed by esterification of linolenate is found only in the portion of the pool available for exchange, whereas that formed by esterification of linoleate and probably palmitate is distributed throughout the entire lecithin pool. The fractional exchange rate of [32P]lecithin of isolated erythrocyte membranes was 3 times that of the lecithin of intact cells. However, the difference in fractional exchange rates between [3H]palmityl-lecithin or [14C]linoleyl-lecithin and [32P]lecithin is reversed in the membranes. These observations suggest that a portion of the total cellular lecithin pool becomes available for exchange only upon disruption of the erythrocyte, and may be located in a relatively inaccessible portion of the membrane such as the inner surface.