Several subtypes of voltage-dependent calcium channels (VDCCs) are present in the presynaptic terminals. In the mammalian hippocampus, P/Q-, N-, and R- but not L-type VDCCs are involved in the fast transmitter release from large mossy fiber (MF) boutons, which are associated with CA3 pyramidal cell dendrites. We investigated whether L-type VDCCs are indeed absent in these large MF boutons. With the use of Sr2+ as the Ca2+ substitute, the stimulus-evoked Sr2+ increment (delta[Sr2+]pre) was evaluated fluorometrically. Delta[Sr2+]pre appeared to be proportional to Sr2+ inflow through VDCCs and was specifically attenuated by conventional VDCC subtype-selective antagonists. The P/Q-type selective omega-agatoxin IVA (AgTx(IVA)) blocked delta[Sr2+]pre with an IC50 of 28 nM and by 30-35% at its maximum effective concentration of 0.5 microM. The N-type selective omega-conotoxin GVIA (CgTx(GVIA)) blocked delta[Sr2+]pre with an IC50 of 15 nM and by 20-25% at its maximum effective concentration of 1 microM. The R-type selective SNX-482 blocked delta[Sr2+]pre with an IC50 of 79 nM and by 20-25% at its maximum effective concentration of 1 microM. The effects of these toxins did not overlap at their maximum effective concentrations and about 70-80% of delta[Sr +]pre was blocked by the simultaneous exposure to these toxins. delta[Sr2+]pre component that is resistant to AgTx(IVA), CgTx(IVA), and SNX-482 was significantly potentiated by an L-type agonist, (S)-(-)-Bay K8644, and attenuated by an L-type antagonist, nimodipine, suggesting that L-type VDCCs are present in large MF terminals. The L-type agonist, (+/-)-Bay K8644, also potentiated Sr2+ inflow into individual boutons identified as large MF boutons under confocal microscopy. Almost similar results were observed for Ca2+ inflow-dependent fluorescence increments. L-type VDCCs appear to be present in large MF boutons and mediate a substantial Ca2+ inflow into presynaptic terminals during action potentials.