A novel dopamine receptor mRNA transcript has been identified and classified as the D3 receptor subtype. We have examined the binding of the D2/D3-selective compound [125I]-Iodosulpride using unlabeled D3-selective 7-OH-DPAT to determine the distribution of D2 and D3 dopamine receptor subtypes in the rat basal forebrain. Use of [125I]-labeled ligands has significant advantages over [3H]-labeled compounds for autoradiographic studies, especially for evaluating small brain areas containing low receptor densities. [125I]-Iodosulpride identified two sites with high affinity (< 1 nM) in the presence of (-)sulpiride (1 microM; D2+3) or 7-OH-DPAT (10 nM; D3), with a greater density of D2 receptors (twofold) compared to D3 receptors in the striatum. The density of D2 and D3 receptor subtypes displayed a 1:1 ratio in the nucleus accumbens. [125I]-Iodosulpride with 7-OH-DPAT displayed D2 sites, predominately in the striatum. Digital subtraction autoradiography showed the highest levels of D3 binding in the islands of Calleja as well as in the core and shell regions of the nucleus accumbens. In sum, the advantages in using [125I]-Iodosulpride to label the dopamine receptor subtypes are high specific activity, affinity, and lack of quenching in autoradiographic analyses. Moreover, masking D3 receptors with 7-OH-DPAT permitted indirect determination of D3 receptor density and localization using the [125I]-labeled ligand, without the potential confound of 7-OH-DPAT binding to sigma receptors. The colocalization of the D2 dopamine receptors with D3 receptors suggests that unique interactions may exist between the receptor subtypes in the rat basal forebrain region.