The effects of testosterone treatment on cholesterol levels and its fatty acid components were studied in adult rats. Cholesterol levels increased both in the liver and in the serum of castrated rats. Androgen administration restored the normal values only in the serum. A general decrease in unsaturated and essential fatty acids in cholesterol esters was evident after testosterone administration. In the liver, only the C16:O/C16:1 ratio clearly increased after testosterone administration, which inhibited the delta 9 unsaturation of palmitic acid, but not of stearic acid. In the serum the C16:O/C16:1, C18:O/C18:1, and C18:2/C2O:4 ratios decreased after castration and were restored by testosterone. The results indicate a clear inhibition of delta 9 unsaturation of palmitic and stearic acids, of delta 5 unsaturation and elongation in organs other than the liver. No effect was evident on delta 6 unsaturation. This suggests that fatty acid unsaturations are regulated differently by testosterone in different tissues. For delta 9 unsaturation in the liver, the effect also seems to be substrate-dependent.