The effect of dietary fat on the distribution of fatty acids within the bilayer of red cell membrane phosphatidylcholine was determined after feeding young female rats semipurified diets containing 10% of either hydrogenated cottonseed oil, menhaden oil, or corn oil for 4 days. Changes in fatty acyl composition of both plasma and erythrocyte phosphatidylcholine were noted. The composition of the fatty acids in phosphatidylcholine in the outer leaflet of the bilayer was determined after hydrolysis of outer leaflet phospholipids, first with phospholipase A2 and subsequently with sphingomyelinase. The distributions of acyl constituents of phosphatidylcholine in the inner and the outer monolayers of the membrane were found to be distinctively different, regardless of the dietary fat intake. For example, palmitic acid and oleic acid were predominantly present in the phosphatidylcholine of the inner monolayer, and arachidonic acid and docosahexaenoic acid were present in that of the outer monolayer. Despite such diet-induced changes in phosphatidylcholine fatty acid composition, no alteration in erythrocyte integrity was observed when monitored by either morphology or ektacytometry.