We have studied by in situ hybridization for GAD65 mRNA in thick sections and by semiquantitative postembedding immunocytochemistry in consecutive semithin sections, the expression of gamma-aminobutyric acid (GABA) and glycine in cell bodies and axosomatic puncta of the rat ventral nucleus of the lateral lemniscus (VNLL), a prominent monaural brainstem auditory structure. The in situ hybridization and the densitometric analysis of the immunostaining suggest that the rat VNLL contains two main populations of neurons. Approximately one-third of neurons are unstained with either technique and are presumably excitatory; their cell bodies are enveloped by a large number of glycine-immunoreactive puncta. Most if not all of the remaining two-thirds colocalize GABA and glycine and are assumed to be inhibitory. These two populations show a complementary distribution within the VNLL, with inhibitory neurons located mainly ventrally and excitatory neurons dorsally. In scatterplots of gray values measured from cell bodies, the double-labeled cells appear to form a single cluster in terms of their staining intensities for the two transmitter candidates. However, this cluster may have to be further subdivided because cells with extreme GABA/glycine ratios differ from those with average ratios with respect to location or size. The VNLL seems unique among auditory structures by its large number of neurons that colocalize GABA and glycine. Although the functional significance of this colocalization remains unknown, our results suggest that the VNLL exerts convergent excitatory and inhibitory influences over the inferior colliculus, which may underlie the timing processing in the auditory midbrain.