PCOS, characterized by hyperandrogenism and chronic anovulation, is frequently associated with insulin resistance. Ample evidence implicates a role for insulin in the genesis of ovarian hyperandrogenism. The objective of this study was to begin to define the intracellular signaling pathway(s) that mediate insulin regulation of 17-hydroxylase activity in human ovarian theca cells. Third passage theca cells isolated from the ovaries of regularly cycling premenopausal women were used. Insulin alone had no effect on 17-hydroxylase activity or CYP17 mRNA expression but required co-stimulation with forskolin. At the insulin concentration used (10 ng/ml), a neutralizing antibody to the insulin receptor but not an antibody to the type I IGF receptor blocked the insulin stimulation of 17-hydroxylase activity, demonstrating that the effects were mediated by the insulin receptor. Insulin stimulated both PI3-kinase and ERK 1/2 (MAP kinase) pathways. Specific inhibition of MEK with PD98059 or I0126 did not decrease the 17-hydroxylase activity stimulated by forskolin or forskolin plus insulin. In contrast, the PI3-kinase inhibitor LY294002 completely blocked insulin-stimulated 17-hydroxylase activity. Our data demonstrate that insulin stimulates PI3-kinase and ERK 1/2 activities in human theca cells but only PI3-kinase mediates the insulin augmentation of forskolin-stimulated 17-hydroxylase activity.