In this study we characterized the distribution of glycine receptor immunoreactivity in the spinal cord of the rat by using monoclonal antisera directed against the purified glycine receptor. There was dense, punctate glycine receptor immunoreactive staining in all regions of the gray matter ventral to the substantia gelatinosa. The densest staining was found in laminae III and IV of the dorsal horn. There were also distinct, tributarylike bands of punctate staining that extended well into the white matter of the lateral and ventral funiculi. The only consistent cell body staining was found in small neurons of the ventral horn. The labelled neurons were distributed among larger, unlabelled motoneurons. In general, the pattern of glycine receptor immunoreactivity was similar at all levels of the spinal cord and was comparable to that seen with binding of a tritiated glycine receptor antagonist, strychnine, to sections of rat spinal cord (Zarbin et al.: J. Neurosci. 1:532-547, '81). Two important exceptions, however, were observed. In contrast to the high levels of strychnine binding reported in the substantia gelatinosa, we found almost no glycine receptor immunoreactivity in laminae I and II of the superficial dorsal horn of the spinal cord or of the trigeminal nucleus caudalis. There was also a notable absence of antibody staining in the intermediolateral cell column of the thoracic cord. The presence of dense glycine receptor immunoreactivity in the region of lamina V and its absence in the superficial dorsal horn are discussed in terms of a possible differential glycinergic control of nociceptive neurons of laminae I and V.