FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. The present studies were performed to determine the actions of FSH and IGF-I on PI3K/AKT protein kinase signaling in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were prepared from 10-d-old rats. After 7 d in culture, Sertoli cells were treated with IGF-I, FSH, or IGF-I plus FSH. In some experiments cultures were treated with 8-bromo-cAMP (40 μm), (Bu)₂cAMP (40 μm), or forskolin (10 μm). After treatments, cell lysates were prepared, and the activation state of AKT and cAMP response element-binding protein (CREB) was determined by Western blot analysis using phosphorylation site-specific antibodies. IGF-I had little effect on CREB phosphorylation, but rapidly increased the phosphorylation of AKT in a concentration-dependent manner. Maximal stimulatory effects of IGF-I were observed at 10–20 ng/ml. Treatment with FSH (0.9 IU/ml) or forskolin for 20 min increased CREB phosphorylation, but had little effect on AKT phosphorylation. However, FSH caused a concentration-dependent increase in IGF-I-induced AKT phosphorylation. Longer incubations (1–4 h) with FSH alone resulted in the elevation of AKT phosphorylation concomitant with an increased secretion of IGF-I and decreased production of IGF-binding protein-3, implicating endogenous IGF-I in the action of FSH on AKT phosphorylation. IGF-I- and FSH-dependent AKT phosphorylation was inhibited by LY29400 (10 μm), a PI3K inhibitor, and by IGF-binding protein 3, but not by a PKA inhibitor (H89). The present study demonstrates that immature rat Sertoli cells possess multiple protein kinase signaling cascades that are regulated by FSH. Furthermore, FSH amplifies IGF-I-mediated PI3K/AKT signaling in Sertoli cells. The results provide evidence for intracellular signaling mechanisms that may be required for the proliferation and differentiation of Sertoli cells.