Cross-reactions between dopamine D3 and sigma receptor ligands were investigated using (+/-)-7-hydroxy-N,N-di-n-[3H]propyl-2-aminotetralin [(+/-)-7-OH-[3H]-DPAT], a putative D3-selective radioligand, in conjunction with the unlabeled sigma ligands 1,3-di(2-tolyl)guanidine (DTG), carbetapentane, and R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [R(-)-PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D3 receptor, neither DTG nor carbetapentane (0.1 microM) displaced (+/-)-7-OH-[3H]DPAT binding. R(-)-PPAP (0.1 microM) displaced 39.6 +/- 1.0% of total (+/-)-7-OH-[3H]DPAT binding. In striatal and nucleus accumbens homogenates, (+/-)-7-OH-[3H]DPAT labeled a single site (15-20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high- and low-affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(-)-PPAP identified two sites in equal proportion. Carbetapentane and R(-)-PPAP (0.1 microM) displaced approximately 20-50% of total (+/-)-7-OH-[3H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)-7-OH-[3H]DPAT binding to sigma receptors, saturation analysis was performed in the cerebellum while masking D3 receptors with 1 microM dopamine. Under these conditions (+)-7-OH-[3H]DPAT labeled sigma receptors with an affinity of 24 nM. These results suggest that (a) (+/-)-7-OH-[3H]DPAT binds D3 receptors with high affinity in rat brain and (b) a significant proportion of (+/-)-7-OH-[3H]DPAT binding consists of sigma 1 sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens.