We describe an in vitro system of swine granulosa cells which remain responsive to estradiol (E2) and FSH. In this system, we examined mechanisms by which E2 amplifies the stimulatory actions of FSH. E2 stimulated progesterone production in a dose-dependent manner and enhanced the tropic effects of FSH. The facilitative interaction between E2 and FSH could not be accounted for by mitogenic effects, by a leftward shift in the dose-response curves for FSH or E2, or by catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. E3 also enhanced stimulatory actions of 8-bromo-cAMP, choleratoxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Lipoproteins were required for maximal interactive effects between E2 and FSH. However, estrogen significantly increased the effects of FSH even in serum- and lipoprotein-free medium. In addition, when de novo cholesterol biosynthesis by granulosa cells was suppressed by Compactin, the actions of E2 and/or FSH were not impeded. In contrast, E2 alone and FSH alone significantly augmented progesterone production in response to the oxygenated sterol 5-cholesten-3 beta, 25-diol, which is an effective substrate for cholesterol side-chain cleavage. In the presence of 5-cholesten-3 beta, 25-diol, the magnitude of the synergism between E2 and FSH was also increased markedly. We conclude that E2 augments the stimulatory actions of FSH or cAMP on steroidogenesis. This facilitative interaction is amplified by exogenous lipoproteins and does not seem to depend upon endogenous cholesterol biosynthesis. Studies with 5-cholesten-3 beta, 25-diol suggest that estrogen and FSH exert significant stimulatory effects at the level of cholesterol side-chain cleavage. These observations reveal important interactions of E2 and fSH in preparing granulosa cells for the high rates of steroidogenesis they ultimately express in the luteinized state.