Two cell lines were created by transfecting cDNAs of the human D2 receptor or the recently cloned human D3 receptor to CHO cells, and the properties of [125I]iodosulpride binding to membranes of these cells were compared. In cell lines expressing the D2 receptor subtype where the selectable marker, a phleomycin-resistance gene, was cotransfected in a different plasmid, a stable expression could be maintained for only few passages. In cell lines expressing the D3 receptor subtype, the selectable marker, a dihydrofolate reductase gene, was cotransfected in the same plasmid and a stable expression could be obtained. In addition, the D3 receptor gene could be amplified in these latter cell lines and a high expression level reached (up to 10(6) binding sites per cell). Sodium and, to a lesser extent, lithium similarly increased [125I]iodosulpride binding to D2 and D3 receptors. In the absence of guanylnucleotide, dopamine had a 24-fold higher apparent affinity at D3 than at D2 receptors. Gpp(NH)p induced rightward shift and steepening of dopamine competition curves at either subtype but the effects were more marked at D2 than at D3 receptors. Several agonists and antagonists, previously regarded as autoreceptor-selective, displayed higher affinities at D3 than at D2 receptors. Although most antagonists used as antipsychotics displayed high affinities at the D3 receptor, all were more potent at the D2 receptor. However, the ratio of Ki values varied over about 10-fold among these compounds, suggesting that they realize differential dopamine receptor subtype occupancy during treatments and that this might be reflected in their clinical profile.