We have addressed the role of Ca2+ channels in mossy fiber synaptic transmission and long-term potentiation (LTP). Whereas the induction of mossy fiber LTP is entirely normal when synaptic transmission is blocked by the glutamate receptor antagonist kynurenate, LTP is blocked in the absence of extracellular Ca2+. These findings suggest that presynaptic Ca2+ entry is essential for mossy fiber LTP. Therefore, the role of different types of presynaptic Ca2+ channels in synaptic transmission and LTP was investigated. Mossy fiber responses were little affected by the L-type Ca2+ channel blocker nifedipine. They were blocked partially by omega-conotoxin-GVIA (N-type) and almost entirely by omega-agatoxin-IVA (P-type). None of these antagonists blocked mossy fiber LTP, nor was its expression associated with a change in sensitivity of synaptic transmission to either of the two toxins. These results, together with previous findings, suggest that the induction of mossy fiber LTP is critically dependent on the entry of Ca2+ into the presynaptic terminal to trigger a series of steps resulting in the long lasting enhancement of evoked glutamate release. Whereas P-type Ca2+ channels are of primary importance in mossy fiber synaptic transmission, both the induction and expression of mossy fiber LTP can occur in the absence of P-type (or N-type) Ca2+ channels.