Synaptic transmission between hippocampal mossy fibers (MFs) and CA3 pyramidal cells exhibits remarkable use-dependent plasticity. The underlying presynaptic mechanisms, however, remain poorly understood. Here, we have used fluorescent Ca2+ indicators Fluo-4, Fluo-5F, and Oregon Green BAPTA-1 to investigate Ca2+ dynamics in individual giant MF boutons (MFBs) in area CA3 traced from the somata of granule cells held in whole-cell mode. In an individual MFB, a single action potential induces a brief peak of free Ca2+ (estimated in the range of 8-9 microm) followed by an elevation to approximately 320 nm, which slowly decays to its resting level of approximately 110 nm. Changes in the somatic membrane potential influence presynaptic Ca2+ entry at proximal MFBs in the hilus. This influence decays with distance along the axon, with a length constant of approximately 200 microm. In giant MFBs in CA3, progressive saturation of endogenous Ca2+ buffers during repetitive spiking amplifies rapid Ca2+ peaks and the residual Ca2+ severalfold, suggesting a causal link to synaptic facilitation. We find that internal Ca2+ stores contribute to maintaining the low resting Ca2+ providing approximately 22% of the buffering/extrusion capacity of giant MFBs. Rapid Ca2+ release from stores represents up to 20% of the presynaptic Ca2+ transient evoked by a brief train of action potentials. The results identify the main components of presynaptic Ca2+ dynamics at this important cortical synapse.