GABAA receptor-mediated presynaptic depolarization is believed to induce presynaptic inhibition of excitatory synaptic transmission. We report here the functional roles of presynaptic GABAA receptors in glycinergic transmission of the rat spinal cord. In mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with native glycinergic and GABAergic nerve terminals, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were isolated from a mixture of both glycinergic and GABAergic sIPSCs by perfusing the SDCN nerve cell body with ATP-free internal solution. Under such experimental conditions, exogenously applied muscimol (0.5 μM) depolarized glycinergic presynaptic nerve terminals and significantly increased glycinergic sIPSC frequency to 542.7 ± 47.3 % of the control without affecting the mean current amplitude. The facilitatory effect of muscimol on sIPSC frequency was completely blocked by bicuculline (10 μM) or SR95531 (10 μM), selective GABAA receptor antagonists. This muscimol-induced presynaptic depolarization was due to a higher intraterminal Cl− concentration, which is maintained by a bumetanide-sensitive Na-K-Cl cotransporter. On the contrary, when electrically evoked, this muscimol-induced presynaptic depolarization was found to decrease the action potential-dependent glycine release evoked by focal stimulation of a single terminal. The results suggest that GABAA receptor-mediated presynaptic depolarization has two functional roles: (1) presynaptic inhibition of action potential-driven glycinergic transmission, and (2) presynaptic facilitation of spontaneous glycinergic transmission.