In contrast to dopamine D1 receptors, the anatomical distribution of D5 receptors in the CNS is poorly described. Therefore, we have studied the localization of dopamine D5 receptors in the brain of rat and human using our newly prepared subtype-specific antibody. Western blot analysis of brain tissues and membranes of cDNA transfected cells, and immunoprecipitation of brain dopamine receptors suggest that this antibody is highly selective for native dopamine D5 receptors. The D5 antibody labeled dopaminergic neurons of mesencephalon, and cortical and subcortical structures. In neostriatum, the D5 receptors were localized in the medium spiny neurons and large cholinergic interneurons. The D5 labeling in caudate nucleus was predominantly in spines of the projection neurons that were frequently making asymmetric synapses. Occasionally, the D5 receptors were also found at the symmetric synapses. Within the cerebral cortex and hippocampus, D5 antibody labeling was prominent in the pyramidal cells and their dendrites. Dopamine D5 receptors were also prominent in the cerebellum, where dopamine innervation is known to be very modest. Differences in the localization of D5 receptors between both species were generally indistinguishable except in hippocampus. In rat, the hippocampal D5 receptor was concentrated in the cell body, whereas in human it was also associated with dendrites. These results show that D5 receptors are localized in the substantia nigra-pars compacta, hypothalamus, striatum, cerebral cortex, nucleus accumbens and olfactory tubercle. Furthermore, the presence of D5 receptors in the areas of dopamine pathways suggests that this receptor may participate actively in dopaminergic neurotransmission.