Polyclonal antisera have been generated against two unique polypeptide fragments in the rat D1B dopamine (DA) receptor, as deduced from the cDNA sequence. Antisera titers were monitored using solid-phase ELISA. Once the titers were established, antisera specificity was determined using Chinese Hamster ovary (CHO) cells, stably transfected with the full-length cDNA for the rat D1B DA receptor. Immunoreactivity following staining with either anti-D1B DA receptor antisera was equivalent, selective for the D1B DA receptor-transfected CHO cells, and expressed at their membrane and within the cell cytoplasm. Minimal immunofluorescent staining for D1B DA receptor proteins was detected in untransfected CHO cells, or in D1A DA receptor-transfected CHO cells. The regional and cellular distribution patterns for the D1B DA receptor subtype were examined in various brain areas and illustrated significant protein levels within the frontal and parietal cortices and in the hippocampus and dentate gyrus. Lesser amounts of receptor protein staining were seen in the dorsal striatum, olfactory tubercle, and cerebellar vermis. D1B DA receptor protein staining was correlated with the cellular expression of D1B DA receptor mRNA transcripts in these same brain regions using concurrent fluorescent analyses. The homologous coincidence in staining patterns for the D1B DA receptor transcripts and encoded proteins in identified neurons of the frontal cortex and striatum showed variations in receptor expression in these identified basal ganglia pathways.