Insulin administration has previously been shown to reverse the effects of chemically-induced and spontaneous diabetes on hepatic drug and steroid metabolism in the rat. The complex network of the intact hormonal system of the body and its physiological feedback mechanisms makes it difficult to ascribe the effects seen to any particular hormones. The present study investigated the effect of insulin on hepatic steroid metabolism in the absence of other hormonal influences by using isolated rat liver cells. Insulin (10(-9) M) produced two peaks of increased enzyme activity in the hepatocytes (at 1/2 hr and 24 hr). Dose-response curves at 1/2 hr and 24 hr insulin preincubation suggest that these two peaks are probably generated by different mechanisms. The absence of any significant changes in cytochrome P-450 content after 1/2, 1 and 2 hr of insulin treatment indicates that the increase in steroid metabolizing enzyme activities is not due to an increase in de-novo enzyme synthesis. Our observations provide further evidence for the role played by insulin in the regulation of hepatic steroid and drug metabolism in the rat.