The products of desaturation and elongation of [1-C-14]18:3(n - 3) and [1-C-14]20:5(n - 3) were studied using hepatocytes and microsomes prepared from livers of trout maintained on diets containing either olive oil or fish oil, to establish the extent to which the formation of 22:6(n - 3) was enhanced in the absence of dietary 22:6(n - 3) and to investigate the pathway(s) of conversion of 18:3(n - 3) and 20:5(n - 3) to 22:6(n - 3). Levels of 20:5(n - 3) and 22:6(n - 3) in the total lipid of hepatocytes from trout fed olive oil were 20-fold and 10-fold, respectively, lower than in cells from trout fed fish oil. For both dietary groups, [1-C-14]18:3(n - 3) was incorporated into hepatocyte lipid to a greater extent than [1-C-14]20:5(n - 3). Almost 70% of the total radioactivity from [1-C-14]18:3(n - 3) was recovered in hepatocyte triacylglycerols, whereas radioactivity from [1-C-14]20:5(n - 3) was recovered almost equally in neutral lipids (52%) and polar lipids (48%). The products of desaturation and elongation from both labelled substrates were esterified mainly into hepatocyte polar lipids, whereas elongation products of [1-C-14]18:3(n - 3) were preferentially incorporated into neutral lipids. Radioactivity recovered in the 22:6(n - 3) of polar lipids of hepatocytes from trout fed olive oil, from both C-14 substrates, was approximately double that in hepatocytes from trout fed fish oil. No radioactivity from either [1-C-14]18:3(n - 3) or [1-C-14]20:5(n - 3) was incorporated into 22:6(n - 3) by microsomes isolated from livers from either group of fish and incubated in the presence of acetyl-CoA, malonyl-CoA, NADH, NADPH, ATP and coenzyme A. However, significant radioactivity was recovered in 24:5(n - 3) and 24:6(n - 3) from [1-C-14]20:5(n - 3) and more radioactive 24:6(n - 3) accumulated in microsomes from trout fed olive oil than from trout fed fish oil. The results establish that the formation of 22:6(n - 3) from both 18:3(n - 3) and 20:5(n - 3) in hepatocytes of rainbow trout is stimulated by omitting 22:6(n - 3) from the diet and are consistent with the biosynthesis of 22:6(n - 3) in trout liver cells proceeding via 24:5(n - 3) and 24:6(n - 3) intermediates.