The synthesis of androgens by theca-interstitial cells is stimulated by LH and the insulin/insulin-like growth factor I (IGF-I) system. An essential element of the steroidogenesis is the uptake of plasma cholesterol and transformation to steroid hormones. In the rat, the uptake of cholesterol by the theca-interstitial cells is mediated by the high density lipoprotein receptor. The goal of the present study was to examine whether insulin has any effect on cholesterol delivery into theca-interstitial cells. The effects of insulin and hCG on the expression of the high density lipoprotein receptor (SR-BI) messenger RNA (mRNA) and intracellular cholesterol levels were examined in rat theca-interstitial cells under in vivo and in vitro conditions. Twenty-five-day-old rats were treated with insulin, hCG, or insulin followed by hCG. The expression of SR-BI mRNA was then examined in ovaries enriched in theca-interstitial cell population by Northern blot analysis. Treatment with insulin increased the expression of SR-BI mRNA over that in controls treated with saline. hCG administration also increased the expression of SR-BI mRNA. A combination of insulin followed by hCG produced an even greater increase in SR-BI mRNA expression. Measurements of cellular cholesterol in the ovarian tissue showed an increase in total and free cholesterol levels in response to insulin treatment. As expected, administration of hCG produced a depletion of cellular cholesterol, and the depletion was even more pronounced in response to treatment with insulin and hCG. The effect of insulin and hCG on SR-SBI mRNA expression was then examined under in vitro conditions using primary cultures of theca-interstitial cells. Treatment with insulin produced an increase in SR-BI mRNA expression. As the cultured theca-interstitial cells were not able to maintain hCG receptors, hCG addition produced no increase in SR-BI mRNA expression. However, in the presence of insulin, these cells were able to maintain hCG receptors and readily responded to hCG to increase SR-BI mRNA expression. Although insulin alone produced a modest increase in total and free cholesterol levels, in the presence of insulin, hCG produced the expected depletion of cellular cholesterol content. The present study shows that insulin has a stimulatory effect on the expression of high density lipoprotein receptors in theca-interstitial cells, suggesting that one of the actions of insulin is to increase intracellular cholesterol, which is subsequently mobilized for androgen biosynthesis in theca-interstitial cells.