Rats were fed diets in which the sole source of fat was either ethyl oleate, linoleate, linolenate or an equal mixture of ethyl linoleate and linolenate. The fatty acid composition of individual phospholipids from platelets and liver was compared to define how total body metabolism regulates which unsaturated fatty acids are produced and incorporated into platelet lipids for potential release and conversion to eicosanoids. The level of 20:4(n-6) in all phospholipids was not markedly altered by feeding linoleate versus that found in chow-fed controls. In oleate fed rats, the 20:3(n-9)/20:4(n-6) ratio varied from 0.5 in liver PE to 4.1 for liver PI, while ratios of 1.0, 1.1, 0.7 and 1.3 were found respectively for platelet PE, PC, PS and PI. Platelet PE contained a component tentatively identified as 22:3(n-9), which is consistent with the finding that this lipid contains significant amounts of 22:4(n-6) and 22:5(n-3) when rats received respectively linoleate or linolenate. Rats fed linolenate have a tight coupling between the regulation of unsaturated fatty acid biosynthesis and the selective acylation of 20:5(n-3) into all lipids. The 20:5(n-3)/20:4(n-6) ratio, however, varied between lipids. In liver PE, PC, PS and PI it was respectively 4.3, 4.9, 3.8 and 0.4, while in the analogous platelet lipids it was 3.0, 4.0, 0.9 and 0.6. Feeding linolenate did not markedly elevate the levels of 22:5(n-3) or 22:6(n-3) in platelet PI, but the combined amounts of 22:5(n-3) and 22:6(n-3) in liver PI were 21.2%, versus 2.9% in chow-fed controls. When the diet contained linoleate and linolenate, there was selective conversion of 18:2(n-6) to 20:4(n-6) and its acylation into lipids versus analogous metabolism of 18:3(n-3) to 20:5(n-3) and its subsequent incorporation. Again, the 20:5(n-3)/20:4(n-6) ratio was lowest for platelet PI and PS and liver PI. Washed human platelets readily incorporated 20:3(n-9), 20:4(n-6) and 20:5(n-3) into phospholipids. With each substrate, PI had the highest specific activity; this effect was most pronounced with 20:3(n-9). These incorporation studies are consistent with the feeding studies which show that oleate is converted to 20:3(n-9) and incorporated into PI more readily than the analogous metabolism of 18:3(n-3) to 20:5(n-3) and its acylation into PI, which is an important source of unsaturated fatty acids for prostaglandin biosynthesis.