Potential cellular substrates for functions ascribed to the dopamine D2 receptor were examined in rat brain using immunoperoxidase for localization of a D2 receptor peptide and immunogold staining for the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). Specificity of the rat polyclonal antiserum, raised against a 15 amino acid fragment from the third intracellular loop of the D2 receptor, was shown by immunoblot analysis and by selective labeling of cultured Chinese hamster ovary cells permanently transfected with the cDNA for the D2 receptor. Although the light microscopic distribution of immunolabeling for the D2 peptide was diffuse, it was selectively localized to regions containing dopamine cells (substantia nigra and ventral tegmental area) or their forebrain projections (dorsal and ventral striatum, nucleus accumbens, and olfactory tubercles). Electron microscopic examination of the medial substantia nigra and ventral tegmental area revealed readily detectable peroxidase immunoreactivity for the D2 peptide, primarily associated with the smooth endoplasmic reticulum and plasmalemmal surfaces of dendrites. Many D2 peptide- immunoreactive dendrites also contained immunogold labeling for TH, although some dendrites were singly labeled for either marker. In the medial and dorsolateral striatum, immunoperoxidase product for the D2 peptide was localized most extensively in dendrites, with the greatest intensity of immunolabeling seen in spines. A number of striatal dendrites exhibiting D2 peptide labeling were contacted by axon terminals immunoreactive for TH. Additionally, D2 peptide immunoreactivity was distributed to some synaptic vesicles and portions of the plasmalemmal surface in unmyelinated axons and in axon terminals. Most D2 peptide-immunoreactive terminals either lacked detectable membrane specializations, or formed thin, symmetric synapses in single sections. A few D2 peptide-labeled terminals formed asymmetric junctions on dendritic spines. In dually labeled sections, most D2 peptide-immunoreactive terminals lacked detectable immunolabeling for TH. However, in fortunate planes of section, peroxidase product for D2 peptide immunoreactivity was occasionally seen in pre-terminal portions of axons whose terminal varicosities contained immunogold labeling for TH. These ultrastructural results are consistent with the localization of a dopamine D2 receptor-like protein that is strategically positioned to subserve (1) autoreceptor functions at the level of dendrites in the midbrain and presynaptic axon terminals in the striatum, as well as (2) postsynaptic actions on striatal spiny dendrites and other nondopamine terminals.