Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]-N-methylpyridinium (ASP(+)). ASP(+) uptake was electrogenic and inhibited by TPA(+) (EC(50) = 2.7 microM), tetraethylammonium (EC(50) = 35 microM), cimetidine (EC(50) = 36 microM), or quinine (EC(50) = 6.7 microM). Stimulation with carbachol or ATP decreased initial uptake by 44 +/- 3 (n = 14) and 34 +/- 4% (n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 +/- 4% (n = 40). Inhibition of calmodulin (CaM), Ca(2+)/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 +/- 2 (n = 15), 40 +/- 4 (n = 30), and 31 +/- 4% (n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC(50) value for the inhibition of ASP(+) uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.