The mitogen-activated protein kinase (MAPK) cascade is stimulated by both receptor tyrosine kinases and G protein-coupled receptors. We show that recombinant human dopamine D(3) receptors expressed in Chinese hamster ovary cells transiently activate MAPK via pertussis toxin-sensitive Gi and/or Go proteins. The involvement of D(3) receptors was confirmed by use of the D(3) agonists PD 128,907 and (+)-7-hydroxy-2-dipropylaminotetralin, which mimicked the response to dopamine (DA). Furthermore, haloperidol and the selective D(3) receptor antagonists S 14297 and GR 218,231 attenuated DA-induced MAPK activation; however, when tested alone, S 14297 weakly stimulated MAPK activity, suggesting partial agonist activity. The transduction mechanisms by which hD(3) receptors activate MAPK were explored with specific kinase inhibitors. Genistein and lavendustin A, inhibitors of tyrosine kinase activity, did not reduce DA-induced MAPK activation. In contrast, PD 98059, an inhibitor of MAPK kinase, and Ro 31-8220 and Gö 6983, inhibitors of protein kinase C (PKC), blocked DA-induced MAPK activation. However, MAPK activation was insensitive to PKC down-regulation by phorbol esters, indicating the involvement of an "atypical" PKC. Furthermore, MAPK activation involved phosphatidylinositol 3-kinase inasmuch as its inhibition by LY 294002 and wortmannin reduced DA-induced MAPK activation. In conclusion, this study demonstrates that stimulation of hD(3) receptors activates MAPK. This action is mediated via an atypical isoform of PKC, possibly involving cross-talk with products of phosphatidylinositol 3-kinase activation.