Isolated bovine rod outer segments (ROS) were incubated under different conditions with radiolabeled fatty acid-Coenzyme A (CoA) compounds, fatty acids and phospholipids in order to further investigate the rates, mechanisms and function of phospholipid metabolism within that organelle. ROS contain acyl CoA synthetase, acyl transferase, acyl CoA hydrolase, and phospholipase A activities. Although different radiolabeled fatty acid CoAs were esterified to the major ROS phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine) at the same rate, different free fatty acids were esterified at different rates. There was no correlation between these estimates of in vitro rates of incorporation of fatty acids and the fatty acid composition of ROS phospholipids. Both the deacylation of radiolabeled phospholipids (phospholipase A activity) and the acylation of endogenous phospholipids (acyl transferase activity) were maximally stimulated when ATP, CoA, Mg2+ and Ca2+ were present, and both processes were stimulated by pro-oxidizing conditions and exposure to light. Under phospholipase A-stimulatory conditions, there was preferential hydrolysis of polyenoic fatty acids from endogenous ROS phospholipids. Both the acylation and deacylation reactions were primarily at the sn-2 position of ROS phospholipids.