Elevated cAMP has been shown to unmask agonist activity of antiprogestin/antiglucocorticoid RU486. In our search for cellular target genes induced through this cross-talk mechanism, we identified human insulin receptor substrate-2 (IRS-2), a cytoplasmic signaling molecule that mediates effects of insulin, insulin-like growth factor-1 (IGF-I), and other cytokines by acting as a molecular adaptor between diverse receptor tyrosine kinases and downstream effectors. Our analysis of the regulation of IRS-2 in HeLa cell models shows that synergistic induction of IRS-2 by cAMP and RU486 can be mediated by progesterone receptors (PR) and glucocorticoid receptors (GR) and occurs through a relative slow mechanism that requires ongoing protein synthesis. Importantly, we demonstrate that IRS-2 mRNA is also inducible by progesterone, while glucocorticoid effects are only observed in the presence of cAMP. Up-regulation of IRS-2 by progesterone depends strictly on the presence of PR and occurs through a rapid mechanism, suggesting that it represents a primary transcriptional response. Furthermore, we show that expression of IRS-1, which also binds to receptors of insulin, IGF-I, and cytokines, is unaffected by progesterone. Thus, our results demonstrate that progesterone alters the ratio of IRS-1 and IRS-2 in PR-positive cells and implicate a mechanism through which progesterone can modulate the effects of insulin, IGF-I, and cytokines on cell proliferation, differentiation, and homeostasis.