Polyclonal antibodies were made in rabbits against glycine conjugated to bovine serum albumin with glutaraldehyde and were used for immunocytochemical studies in the cochlear nucleus and superior olivary nucleus of the guinea-pig. Antibodies selective for glycine were prepared by affinity chromatography. By dot-blot analysis this preparation showed a strong recognition of glycine conjugates and relatively little recognition of conjugates of most other amino acids tested. However, there was a significant reaction with conjugates of alanine and beta-alanine, and this cross-reaction could not be removed by affinity chromatography without eliminating the preparation's recognition of glycine. The affinity-purified preparation showed only a weak recognition of conjugates of gamma-aminobutyrate (GABA) which was detectable at high concentrations of primary antibody. Immunocytochemical studies showed several intensely staining cell bodies in the cochlear nucleus and superior olivary complex. Most immunoreactive cell bodies in the cochlear nucleus were in the dorsal cochlear nucleus, being present in both the superficial and deep layers. Scattered immunoreactive cells were present in the ventral cochlear nucleus. Intense staining of cell bodies was seen in the medial nucleus of the trapezoid body, and these cells appear to correspond to the principal cells of that nucleus. Punctate labelling, suggestive of immunoreactive presynaptic terminals, was also apparent, particularly in the ventral cochlear nucleus and lateral superior olive. In the ventral cochlear nucleus, immunoreactive puncta were found around unlabeled cell bodies, at times nearly covering the perimeter of the cell. A population of glycine-immunoreactive cell bodies in the superficial dorsal cochlear nucleus also labeled with anti-GABA antibodies as determined through double-labeling studies. However, glycine-positive cells in the deep dorsal cochlear nucleus were not labeled with anti-GABA antibodies, and some populations of GABA-positive cells in the superficial layers were not labeled with anti-glycine antibodies. In the hippocampus intense staining of cell bodies and puncta was seen with anti-GABA antibodies while essentially no staining was seen with anti-glycine antibodies. These results suggest that anti-glycine antibodies can be useful for immunocytochemical identification of glycinergic neurons. From this study several populations of putative glycinergic neurons are identified in the auditory nuclei of the brain stem using these antibodies. Some populations of GABA-containing neurons also contain high levels of glycine or a related molecule.