The central auditory system contains several predominantly glycine-immunoreactive nuclei, and one of these, the lateral nucleus of the trapezoid body, contains cell bodies exhibiting a spectrum of labeling intensity. By using post-embedding glycine immunocytochemistry on thin sections, and toluidine blue staining of adjacent sections, we established that darkly glycine-immunoreactive neurons constituted a distinct morphological class and form one of three subnuclei of the lateral nucleus of the trapezoid body, called the posteroventral subnucleus. These neurons resemble, in both labeling intensity and cell body morphology, the principal cells of the medial nucleus of the trapezoid body. The other two subnuclei of the lateral nucleus of the trapezoid body, its main and hilus subnuclei, contained predominantly glycine-immunoreactive and glycine-immunonegative neurons, respectively. Glycine immunoreactivity was compared with gamma-aminobutyric acid (GABA) immunoreactivity in order to identify other organizational features of the lateral nucleus of the trapezoid body. Cell bodies that displayed either dark glycine-immunoreactivity or which were glycine-immunonegative were GABA-immunonegative. Cell bodies that displayed GABA immunoreactivity were preferentially located in the main subnucleus. Patterns of distribution of axosomatic innervation in the lateral nucleus of the trapezoid body were revealed in which glycine-immunoreactive puncta were (1) more numerous than GABA-immunoreactive puncta on glycine-immunonegative cell bodies and (2) equal to or less numerous than GABA-immunoreactive puncta on glycine-immunoreactive cell bodies. The characteristics of neural circuitry revealed by glycine and GABA immunoreactivity in the lateral nucleus of the trapezoid body may be generalizable to other populations of neurons of the superior olivary complex and to other regions of the central nervous system containing glycinergic neurons, such as the retina.