FSH is known to activate Gs/cAMP signaling pathway in Sertoli cells (SCs) to support spermatogenesis. However, the molecular mechanism of FSHinduced Gs/cAMP-independent Ca2+-influx in SCs is not clear. In this study, FSH indeed induced an immediate and dose-dependent intracellular Ca 2+-elevation in rat SCs. In the presence of EDTA (2.5 mM) or in the absence of extracellular Ca2+, the FSH-induced intracellular Ca 2+-elevation was abolished. The confocal microscopic observation of Ca2+ image revealed that the SC cellular Ca2+ level was gradually increased after 50 sec of FSH treatment. Dantrolene, a blocker of intracellular Ca2+ release, did not affect this FSH-induced intracellular Ca2+ elevation. The pretreatment of rat SCs with phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, U73122 (3 and 10 μM), inhibited the FSH-induced Ca2+-influx in a dose-dependent manner, but treatment with Gs-specific inhibitor, NF449 (0.1 and 0.3 μM), did not. On the other hand, the activation of Gαh was immediately induced by FSH in the rat SCs within 5 sec of treatment. The translocation of PLC-δ1 from cytosol to cell membrane and the formation of Gαh/PLC-δ1 complexes occurred within 5 and 10 sec, respectively, of FSH exposure. The intracellular inositol 1,4,5-triphosphate (IP3) production was also detected after 30 sec of FSH treatment. The synthetic peptide of PLC-δ1 (TIPWNSLKQGYRHVHLL), not Gs inhibitor, predominantly inhibited the FSH-induced PLC-δ1 translocation, formation of Gαh /PLC-δ1 complex, intracellular IP3 production, and Ca2+ influx. In contrast, the peptide did not interfere with FSH-induced intracellular cAMP accumulation. In conclusion, the FSH-induced immediate Ca2+ influx is unambiguously mediated by an alternative Gαh /PLC-δ1/IP3 pathway that is distinct from the Gs/cAMP pathway in rat SCs.