The acylation of membrane phospholipids in isolated bovine rod outer segments (ROS) by exogenous, radiolabeled palmitic acid (16:0) was assayed, using [14C]palmitoyl-coenzyme A (16:0-CoA), [3H]16:0 alone and [3H]16:0 with added ATP and CoA. Both [14C]16:0-CoA and [3H]16:0 with added ATP and CoA are incorporated predominantly into phosphatidylcholine (PC), whereas [3H]16:0 without added ATP and CoA is incorporated almost exclusively into phosphatidylethanolamine (PE). Without added ATP and CoA in the incubation medium, [3H]16:0 is esterified predominantly to the 2 position of PE, but with added ATP and CoA, it is esterified to both the 1 and 2 positions; the rates of esterification of [3H]16:0 to the 2 position of PE under these two conditions are similar. These results suggest that 16:0 is esterified to the 2 position of PE of ROS membranes by an acyl transferase mechanism that does not utilize an acyl-CoA intermediate. The product of this reaction is likely to be a molecular species of PE with 16:0 at the 2 position and a polyunsaturated fatty acid at the 1 position.