Dopamine D₅ receptor localization has been difficult because even the most specific ligands cannot distinguish between molecular subtypes of the D₁‐like receptor subfamily. Antifusion protein rabbit polyclonal antibodies directed against the C‐terminus of human D₅ receptor were therefore developed for immunolocalization of the D₅ receptor protein in brain. The antibodies were characterized by immunoblot analysis and immunoprecipitation and used for light microscopic immunocytochemistry in rat and monkey brain. Affinity purified D₅ antibodies were specific for D₅ fusion protein as well as cloned and native D₅ receptor on Western blots, and D₅ antisera specifically immunoprecipitated solubilized, cloned D₅ receptor. Regional distribution of D₅ receptor immunoreactivity was consistent across species and correlated well with D₅ mRNA distribution previously reported in monkey brain. Immunoreactivity was widespread and tended to label perikarya and proximal dendrites of neurons in cerebral cortex, basal ganglia, basal forebrain, hippocampus, diencephalon, brainstem, and cerebellum. Neuropil was immunoreactive in olfactory bulb, islands of Calleja, cerebral cortex, superior colliculus, and molecular layer of cerebellum. The distribution of D₅ in brain was clearly different from that of other dopamine receptor subtypes, including D₁, the other member of the D₁‐like receptor subfamily. This unique distribution corroborates the idea that the D₅ receptor subtype has a distinct role in dopamine neurotransmission. Synapse 2:125–145, 2000. © 2000 Wiley‐Liss, Inc.