Anatomical and electrophysiological evidence has raised the possibility that corelease of GABA and glutamate occurs at hippocampal mossy fibre synapses which, however, lack the vesicular GABA transporter VGAT. Here, we apply immunogold cytochemistry to show that GABA, like glutamate, has a close spatial relation to synaptic vesicles in rat mossy fibre terminals, implying that a mechanism exists to package GABA in synaptic vesicles. We also show that GABAA and AMPA receptors are colocalized at mossy fibre synapses. The expression of GABA and GABAA receptors is, however, weaker than in inhibitory synapses. Electrical stimuli that recruit mossy fibres evoke monosynaptic GABAA receptor-mediated signals in post-synaptic targets that show marked frequency-dependent facilitation and sensitivity to group II metabotropic receptors, two features that are characteristic of mossy fibre transmission. These results provide further evidence for GABA and glutamate cotransmission at mossy fibre synapses, although paired pre- and post-synaptic recordings will be required to determine the role of GABA at this unusual synapse.