Our previous study demonstrated that follicle-stimulating hormone (FSH)-induced immediate Ca²⁺-influx in rat Sertoli cells (SCs) is mediated by Gh/phospholipase C-1 (PLC-1) signaling pathway. As to which Ca²⁺-channel is responsible for such Ca²⁺-influx was not understood. In this study, thapsigargin triggered an in-stored calcium release, and evoked a 1.5-fold elevation of intracellular Ca²⁺ in Ca²⁺-free media while FSH exhibited no effect. The re-addition of CaCl₂ (2.5 mM) to FSH-pretreated or thapsigargin-sensitized SCs in Ca²⁺- free media immediately elicited a rapid Ca²⁺-influx or a 2-fold of second intracellular Ca²⁺ elevation, respectively. The addition of Ca²⁺ chelator, EGTA (0.2 mM), reduced the FSH-induced elevation of intracellular Ca²⁺ in SCs incubated with CaCl₂. However, the pretreatment with dantrolene (25 µM), which inhibits in-stored calcium release, did not affect the FSH-induced elevation of intracellular Ca²⁺. NiCl₂ (10 µM), a T-type calcium channel blocker, abolished the FSH-induced SC Ca²⁺-influx. Furthermore, mibefradil (10 and 100 µM), another specific blocker for T-type Ca²⁺-channels, dose-dependently suppressed the FSH-induced Ca²⁺-influx. In contrast, nifedipine (10 and 50 µM) or -conotoxin GVIA (100 and 500 nM), blocker of L- or N-type Ca²⁺-channels, respectively, did not affect the FSH-induced SC Ca²⁺-influx. On the other hand, FSH-induced Ca²⁺-influx was significantly reduced by the pretreatment of SCs with myristoylated synthetic peptide (0.1 and 1 µM) of PLC-1 fragment " TIPWNSLKQGYRHVHLL", but not interfered by 2',5'-dideoxyadenosine (3 and 15 µM), a selective inhibitor of adenylate cyclase. In conclusion, the FSH-induced Gh/PLC-1 pathway-dependent Ca²⁺-influx of rat SCs is mediated by T-type Ca²⁺-channels and independent of instore-calcium release.