The distribution of the D-3 and D-2 dopamine receptor subtypes in forebrain regions of the basal ganglia and mesocorticolimbic system was determined. This was assessed through combined fluorescent visualization of subtype selective anti-peptide antibodies for these cloned receptors and detection of their ligand recognition sites using the D-2 subfamily antagonist, N-(p-aminophenethyl) spiperone (NAPS fluoroprobe). The double-labeling technique enabled direct comparison of the cloned receptor proteins and NAPS fluoroprobe binding in vitro. The application of these two methods together produced results comparable to single-labeling paradigms. Functional D-3 receptors, defined as the coincident fluorescence of the D-3 receptor antisera and fluoroprobe binding, were detected in the core region of the nucleus accumbens and exhibited a laminated expression pattern in the frontal cortex. D-3 receptor protein was expressed robustly in neurons of the dorsolateral striatum, but showed an intense neuropil reaction in the globus pallidus. Functional D-2 receptors, defined as the coincident fluorescence of the D-2 receptor antisera and fluoroprobe binding, were detected in the frontal cortex and the medial shell of the nucleus accumbens. Thus, heterogeneities occurred in the cellular expression of functional D-3 and D-2 receptors in forebrain dopaminoceptive areas. D-3 appears more related to basal ganglia and structures involved with motoric behavior, while D-2 was associated with regions associated with cognitive/affective functions. (C) 1995 Wiley-Liss, Inc.