The function of the D(3) dopamine (DA) receptor remains ambiguous largely because of the lack of selective D(3) receptor ligands. To investigate the function and intracellular signaling of D(3) receptors, we established a PC-12/hD3 clone, which expresses the human D(3) DA receptor in a DA producing cell line. In this model, we find that the D(3) receptor functions as an autoreceptor controlling neurotransmitter secretion. Pre-treatment with 3,6a,11, 14-tetrahydro-9-methoxy-2 methyl-(12H)-isoquino[1,2-b] pyrrolo[3,2-f][1,3] benzoxanzine-1-carboxylic acid, a D(3) receptor preferring agonist, dose-dependently suppressed K+-evoked [3H]DA release in PC-12/hD3 cells but not in the control cell line. This effect was prevented by D(3) receptor preferring antagonists GR103691 and SB277011-A. Furthermore, activation of D(3) receptors significantly inhibits forskolin-induced cAMP accumulation and leads to transient increases in phosphorylation of cyclin-dependent kinase 5 (Cdk5), dopamine and cAMP-regulated phosphoprotein of M(r) 32 000 and Akt. Because we observed differences in Cdk5 phosphorylation as well as Akt phosphorylation after DA stimulation, we probed the ability of Cdk5 and phosphatidylinositol-3 kinase (PI3K) to influence DA release. Cdk5 inhibitors, roscovitine, or olomoucine, but not the PI3K inhibitor wortmannin, blocked the D(3) receptor inhibition of DA release. In a complimentary experiment, over-expression of Cdk5 potentiated D(3) receptor suppression of DA release. Pertussis toxin, 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one and cyclosporine A also attenuated D(3) receptor-mediated inhibition of DA release indicating that this phenomenon acts through Gi/oalpha and casein kinase 1, and phosphatase protein phosphatase 2B (calcineurin), respectively. In support of previous data that D(3) DA receptors reduce transmitter release from nerve terminals, the current results demonstrate that D(3) DA receptors function as autoreceptors to inhibit DA release and that a signaling pathway involving Cdk5 is essential to this regulation.