At least five members of the dopamine receptor family have been characterized at the gene level. D2, D3 and D4 dopamine receptors are related pharmacologically. In order to visualize the differential expression of D1, D2 and D3 receptors in rat brain we have combined in situ hybridization histochemistry with receptor autoradiography. Regions enriched with D3 messenger RNA (mRNA) included the islands of Calleja (ioC) and nucleus accumbens. Very low or undetectable levels were present in the caudate-putamen. In contrast, no D2 transcripts were observed in the islands of Calleja, but there were high levels in the nucleus accumbens, caudate-putamen (CP) and pyramidal layer of the olfactory tubercle. A comparison of the binding pattern of six dopamine receptor radioligands hitherto regarded as D2 receptor-selective showed that the islands of Calleja were intensely labelled by [125I]iodosulpride, [3H]CV 205 502 and [3H]SDZ 205 501, while the binding of [3H]spiperone, [3H]raclopride and [3H]YM 09151-2 was much lower or undetectable. Pharmacological analysis of the binding of D2/D3 ligands to the islands of Calleja and caudate-putamen suggests that binding sites in these two regions are of different pharmacology, consistent with the presence of D3 sites in the islands of Calleja and the predominance of D2 sites in the caudate. These results demonstrate the expression of D3 binding sites in the rat brain and provide a procedure to differentiate D2 and D3 receptor populations in binding studies.