The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D(2) dopamine receptors (D(2)R) has been reported. The high affinity of DA and other DAT substrates for the D(2)R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP(+)) with live cell imaging techniques to identify the role of two D(2)R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D(2)R regulation of DAT. Addition of the D(2)/D(3) receptor agonist quinpirole (0.1-10 muM) to human embryonic kidney cells coexpressing human DAT and D(2) receptor (short splice variant, D(2S)R) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP(+) accumulation. The D(2)/D(3) agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP(+) accumulation. D(2S)R activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP(+) accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D(2)R stimulation. These experiments demonstrate that D(2S)R stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D(2)R. Together, these results suggest a novel mechanism by which D(2S)R autoreceptors may regulate DAT in the central nervous system.