Although FSH receptors are linked to the cAMP second messenger system, additional intracellular signaling pathways appear to be required for the induction of aromatase and the LH receptor during granulosa cell differentiation. We employed adenovirus vectors to modulate specific intracellular signaling systems in undifferentiated granulosa cells to identify the signaling pathway(s) that may be involved in the FSH-mediated induction of aromatase and the LH receptor. Expression of either the constitutively activated human LH receptor D578H or the constitutively active human GS Q227L resulted in increased cAMP production without increasing aromatase activity or mRNA levels for the LH receptor. To explore the contribution of other pathways, we expressed constitutively activated forms MEK and PKB. Neither MEK nor PKB alone increased estrogen or progesterone production by undifferentiated granulosa cells. Stimulation of granulosa cells by FSH in the presence of the constitutively active PKB, but not MEK, let to an amplification of FSH-induced aromatase and LH receptor mRNA levels whereas a dominant negative PKB vector completely abolished the actions of FSH. Expression of the constitutively active PKB in combination with the constitutively active LH receptor D578H, the constitutively active GS Q227L or 8Br-cAMP led to an induction of aromatase as well as the LH receptor mRNA comparable to that seen in cells stimulated with FSH alone. These results demonstrate that PKB is an essential component of the FSH-mediated granulosa cell differentiation and that both the PKB and Gs signaling pathways are required.