We have previously provided functional evidence that glycine and GABA are contained in the same synaptic vesicles and coreleased at the same synapses in lamina I of the rat spinal dorsal horn. However, whereas both glycine receptors (GlyRs) and GABA_Areceptors (GABA_ARs) are expressed on the postsynaptic target, under certain conditions inhibitory events appeared to be mediated by GlyRs only. We therefore wanted to test whether GABA_Breceptors could be activated in conditions where GABA released was insufficient to activate GABA_ARs. Focal stimulation in the vicinity of visually identified lamina I neurons elicited monosynaptic IPSCs in the presence of ionotropic glutamate receptor antagonists. Pairs of stimuli were given at different interstimulus intervals (ISI), ranging from 25 ms to 1 s to study the depression of the second of evoked IPSCs (paired pulse depression; PPD). Maximal PPD of IPSCs was 60 ± 14% (SE) (of the conditioning pulse amplitude), at ISI between 150 and 200 ms. PPD was observed with IPSCs evoked at stimulus intensities where they had no GABA_AR component. PPD of small evoked IPSCs was not affected by the GABA_AR antagonist bicuculline but significantly attenuated by 10–30 μM CGP52432, a specific GABA_Breceptor antagonist. These data indicate that, under conditions where GABA released is insufficient to affect postsynaptic GABA_ARs at lamina I inhibitory synapses, significant activation of presynaptic GABA_Breceptors can occur.