Hippocampal mossy fibres form distinct synapses on principal cells and interneurons. These synapses exhibit distinct forms of use-dependent plasticity, both short- and long-term. The mechanisms underlying this heterogeneity remain poorly understood. An important role in synaptic facilitation has been attributed to presynaptic kainate receptors, which have also been implicated in the initiation and maintenance of epileptiform activity. We have therefore examined the role of presynaptic kainate receptors at distinct presynaptic structures in rat mossy fibres. We applied two-photon microscopy to address Ca²⁺ dynamics in individual mossy fibre boutons (MFBs), which were traced up to 1.5 mm from granule cell somata held in whole-cell mode. In giant MFBs forming synapses on CA3 pyramidal cells, blockade of kainate receptors with 25 μM NBQX increased the amplitude of evoked presynaptic Ca²⁺ transients following an action potential generated in a single cell by 36 ± 14% (p < 0.04, n = 7). Blocking kainate receptors also reduced short-term facilitation of presynaptic Ca²⁺ transients following repetitive spikes (paired-pulse ratio decreased by 52 ± 11%, p < 0.005). Blocking kainate receptors also abolished the contribution of Ca²⁺ stores to the evoked presynaptic Ca²⁺ signal (ryanodine decreased the ΔF/F Ca²⁺ signal under control conditions by 14 ± 3%, n = 10, but only by 3 ± 8%, n = 8, after application of NBQX). In contrast, in en-passant MFBs and in collateral axonal branches, both of which are presynaptic to interneurons, blockade of kainate receptors had no detectable effect on presynaptic Ca²⁺ signalling (n = 7 main axon boutons and n = 8 collateral branches). Consistent with this finding, the contribution of internal Ca²⁺ stores to the transient presynaptic Ca²⁺ signal (in response to a short train of action potentials) was undetectable in en-passant MFBs (average change in ΔF/F after ryanodine application: + 9 ± 9%, n = 9). These data provide evidence at the level of single synapses that presynaptic kainate receptors (a) may act as autoreceptors at granule cell – CA3 pyramidal cell synapses, (b) are functionally expressed in a cell-target dependent manner, and (c) involve activation of Ca²⁺ release from presynaptic Ca2+ stores. Whether this functional diversity can explain distinct features of transmission at the respective synapses is currently under investigation.