Long-term potentiation (LTP) and long-term depression (LTD) are induced presynaptically at the hippocampal mossy fibre-CA3 synapse. Activation of presynaptic metabotropic glutamate receptors (mGluRs) is necessary, but not sufficient for the LTD induction. Using mouse hippocampal slices, we attempted to identify additional presynaptic factors involved in the induction of mossy fibre LTD. Suppression of a rise in the presynaptic intracellular Ca2+ concentration ([Ca2+]i) with a membrane-permeable Ca2+ chelator, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), reduced the magnitude of LTD, whereas an increase in Ca2+ influx induced LTD, suggesting that an elevation of presynaptic [Ca2+]i is crucial for the LTD induction. A broad-spectrum protein kinase inhibitor, H-7, blocked LTD without affecting a presynaptic inhibition induced by an mGluR agonist. Furthermore, LTD was reduced by an inhibitor of calmodulin or Ca2+/calmodulin-dependent protein kinases. Thus, we conclude that mossy fibre LTD requires an increase in presynaptic [Ca2+]i and subsequent activation of Ca2+/calmodulin-dependent protein kinases. Because mossy fibre LTP may also require a rise in presynaptic [Ca2+]i, bidirectional long-term plasticity at the mossy fibre synapse is likely to be regulated by presynaptic Ca2+-dependent processes.