From 25, 15, and 5 C-acclimated green sunfish (Lepomis cyanellus), liver microsomes were prepared by Ca precipitation. During cold acclimation, phosphatidyl choline (PC) declined and phosphatidylethanolamine (PE) and inositol increased. In coldadapted fish the proportion of saturated fatty acids in the phospholipids (PLs) was less, the proportion of monounsaturated fatty acids was greater, and the proportion of polyunsaturated fatty acids did not change. The ratio of saturated to unsaturated fatty acids increased on cold acclimation as previously reported. Microsomes from winter fish contained less saturated fatty acids and more mono- and polyunsaturated fatty acids than microsomes from summer fish when the fish were similarly acclimated in the laboratory. Analyses of incorporation of fatty acid substrates (16:0, 8:0, 18:2n6, 8:3n3) showed that palmitate was preferentially incorporated into phosphatidyl classes. The proportion of labeled palmitic acid in phosphatidic acid may reflect reorganization of the total lipid pool. Incorporation of 14C-labeled stearic acid into PL was greater in PE than in other PLs. Polyunsaturated fatty acids were incorporated into PL fractions in a ratio reflecting their proportions in composition. Microsomal Δ9 desaturation of saturated fatty acids increased in cold acclimation. Coenzyme A derivatives were incorporated more than free acids. Desaturation of polyunsaturated acids by Δ6 desaturase did not increase. Acclimation to reduced temperature results in changes in PL classes and in proportions of fatty acids in microsomes. Δ9 desaturation can account for the increase in monounsaturated fatty acids in cold-acclimated fish.