The effects of insulin on ovarian steroidogenesis were examined in four-day incubations of minced stroma and theca obtained from a woman with hyperandrogenism, insulin resistance, and acanthosis nigricans, and from a normally cycling woman. In incubations of theca obtained from the patient with hyperandrogenism, insulin resistance, and acanthosis nigricans, lutenizing hormone (LH) (25 ng/mL) alone stimulated androstenedione, testosterone, progesterone, and estradiol accumulation. Insulin (500 ng/mL) alone stimulated androstenedione and testosterone accumulation, but not progesterone or estradiol accumulation. In incubations of stroma obtained from the hyperandrogenism, insulin resistance, and acanthosis nigricans patient, LH (25 ng/mL) alone stimulated androstenedione and testosterone accumulation, but not dihydrotestosterone accumulation. Insulin (500 ng/mL) alone stimulated androstenedione, testosterone, and dihydrotestosterone accumulation. In incubations of stroma from the normally cycling woman, LH plus insulin acted synergistically to stimulate androstenedione accumulation. These results suggest that insulin may be a regulator of steroid biosynthesis in the thecal and stromal compartments of the human ovary.