Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]- N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. K_mvalues of hOCT2-mediated uptake of 95 μM amiloride and 24 μM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC₅₀(in μM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca²⁺/CaM complex (−55 ± 5%, n = 10 and −63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (−54 ± 5%, n = 14, and −31 ± 6%, n = 26) and PKA (−16 ± 5%, n = 16, and −18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca²⁺/CaM complex resulted in a significant decrease of V_max(160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (−22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca²⁺/CaM complex causes changes in transport capacity via hOCT2 trafficking.