Presynaptic glycine receptors (GlyRs) have been implicated in the regulation of glutamatergic synaptic transmission. Here, we characterized presynaptic GlyR-mediated currents by patch-clamp recording from mossy fiber boutons (MFBs) in rat hippocampal slices. In MFBs, focal puff-application of glycine-evoked chloride currents that were blocked by the GlyR antagonist strychnine. Their amplitudes declined substantially during postnatal development, from a mean conductance per MFB of ∼600pS in young to ∼130pS in adult animals. Single-channel analysis revealed multiple conductance states between ∼20 and ∼120pS, consistent with expression of both homo- and hetero-oligomeric GlyRs. Accordingly, estimated GlyRs densities varied between 8–17 per young, and 1–3 per adult, MFB. Our results demonstrate that functional presynaptic GlyRs are present on hippocampal mossy fiber terminals and suggest a role of these receptors in the regulation of glutamate release during the development of the mossy fiber – CA3 synapse.