Transfection of a human dopamine D 3 receptor cDNA in a neuroblastoma-glioma hybrid cell line (NG 108-15) provided clonal cell lines stably expressing up to 600 fmol per mg protein of [ 125I]iodosulpiride binding sites. Dopamine and several agonists distinguished two receptor-affinity states in membranes. In the case of dopamine, the high-affinity state ( K i = 0.9 nM, 30% of total binding) was completely conerted into a low-affinity state ( K i = 57 nM) in the presence of 10 μM guanosine-5′- O-(3-thiotriphosphate). In addition to these two sites, a site with a very low affinity for dopamine was evidenced in whole cells. The dopamine D 3 receptor mediated two responses: c- fos activation, as measured by the appearance of Fos-like immunoreactivity, and increased mitogenesis, as measured by incorporation of [ 3H]thymidine. The Fos-like immunoreactivity appeared within 30 min, lasted 2 h and was blocked by the partially selective dopamine D 3 receptor compound (+)-UH 232 (cis-(+)-5-methoxy-1-methyl-2-(di- n-propylamino)tetralin). The mitogenic effect, which occurred after a lag time (over 2 h stimulation), was produced with subnanomolar potency and full intrinsic activity by several compounds previously identified as dopamine D 2 receptor agonists, e.g. quinpirole, (+)-7-OH-DPAT ((+)-7-hydroxy-2-(di- n-propylamino)tetralin) and RU 24926 ( N- n-prophyl-di- β(3-hydroxyphenyl)-ethylamine), and was reversibly blocked by (+)-UH 232 ( K i = 9 nM). Talipexole (B-HT 920, 5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin) was identified as a partial agonist at the dopamine D 3 receptor. Dopamine D 3 receptor-mediated mitogenesis was potentiated by a phorbol ester and was abolished by pretreatment with pertussis toxin. A mitogenic effect of same amplitude was elicited by bradykinin or carbachol, both acting through constitutive receptors. Bradykinin markedly activated inositol phosphate turnover, and had no effect on forskolin-stimulated cyclic AMP accumulation. Carbachol inhibited forskolin-stimulated cyclic AMP accumulation and had no effect on inositol-phosphate turnover. Quinpirole had no effect on any of these second messenger pathways. Thus, in transfected NG 108-15 cells, the dopamine D 3 receptor is coupled to a pertussis toxin-sensitive G protein and mediates two possibly unrelated biological effects, through initial biochemical events that remain to be identified.