During micturition, according to the concept of Blok, Holstege, and colleagues ([1997] Neurosci. Lett. 233:109-112), the pontine micturition center (PMC) elicits bladder contraction by way of direct excitation of the parasympathetic bladder motoneurons. At the same time, the PMC elicits relaxation of the external urethral sphincter (EUS) by excitation of gamma-aminobutyric acid (GABA)-ergic interneurons in the sacral dorsal gray commissure (DGC), which, in turn, inhibit EUS motoneurons. The question is whether the inhibitory neurotransmitter glycine is also involved in this system. The present study investigated, first, whether there are glycine immunoreactive interneurons in the sacral DGC and, second, whether they receive direct PMC afferents. Finally, it was determined whether glycine and GABA are colocalized in DGC interneurons. In two adult male cats, the PMC was identified by electrical stimulation. Subsequently, the identified region was injected with the anterograde tracer WGA-HRP. Sections of sacral cord segments were processed for light and electron microscopic detection of anterograde labeling, as well as for glycine and GABA, using postembedding immunogold labeling with antibodies. In total 128 labeled PMC terminals were found in the DGC, which contained many round vesicles and asymmetric synapses. About 31.3% (40 of 128) made contact with glycine-immunoreactive dendrites. Eleven of them were selected for serial sectioning, which showed that 54.6% (6 of 11) of the glycine-immunoreactive dendrites were also immunoreactive for GABA. The results demonstrate that the PMC projects directly to dendrites of interneurons in the sacral DGC, which are immunoreactive for both glycine and GABA. These interneurons are thought to inhibit the EUS motoneurons during micturition.