Male weanling rats were fed for 28 d a purified diet containing 20% (wt/wt) fat providing high levels of either saturated fat or alpha-linolenic acid or eicosapentaenoic and docosahexaenoic acids with or without 2% (wt/wt) cholesterol supplementation. Effect of diet on rate of desaturation of eicosatrienoic acid (20:3 omega 6) and lipid composition of liver microsomal membranes was examined. The desaturation of 20:3 omega 6 to arachidonic acid (20:4 omega 6) was higher in rats fed linseed oil and lower in rats fed fish oil than in control animals fed the beef tallow diet. The desaturation of 20:3 omega 6 was lower in rats fed beef tallow or linseed oil diets supplemented with cholesterol than in the respective unsupplemented diet. Inclusion of 2% (wt/wt) cholesterol in the fish oil diet failed to affect synthesis of 20:4 omega 6 from 20:3 omega 6. These in vitro changes in delta 5-desaturase activity are consistent with the diet-induced alterations observed in the fatty acid composition of microsomal membranes. Both free cholesterol and cholesterol ester in the microsomal membrane were higher in rats fed beef tallow or linseed oil diets supplemented with exogenous cholesterol than in the respective unsupplemented diet, and only free cholesterol was higher in rats fed the fish oil diet supplemented with cholesterol. Feeding fish oil appears to prevent both the inhibition of 20:4 omega 6 biosynthesis and the accumulation of cholesterol ester that were apparent when 2% cholesterol was added to either beef tallow or linseed oil diets.