Cultured thecal preparations from human ovarian follicles obtained from patients during the follicular phase of the menstrual cycle produced large amounts of delta 4-androstenedione and smaller amounts of testosterone and other androgens when stimulated by hCG, (Bu)2cAMP, and prostaglandin E2, but not when stimulated by FSH. Prostaglandin E2 also stimulated thecal cAMP production under the same conditions. By contrast, androgen production by granulosa cells was negligible with or without gonadotropins in the culture medium. Granulosa cells also did not produce 17 beta-estradiol, even when exposed to FSH and hCG, unless supplied with exogenous delta 4-androstenedione. On the other had, thecal, preparations did not produce significant amounts of 17 beta-estradiol even in the presence of FSH, hCG, and relatively large amounts of exogenous delta 4-androstenedione. These observations suggest that delta 4-androstenedione produced by LH or hCG-stimulated thecal cells is the main substrate for the synthesis of 17 beta-estradiol by FSH-stimulated granulosa cells in vivo.