This study aims to characterize the complex mechanisms of acute regulation of organic cation (OC) transport across the basolateral membrane of freshly isolated male mouse proximal tubules (PTs) using a microtiter plate based fluorescence reader. The fluorescent dye ASP⁺, 4-(-4-(dimethylamino)styryl-N-methylpyridinium, served as substrate for the quantification of OC transport. Wortmannin (WOR, 0.1 mM), an inhibitor of phosphatidylinositol-3-kinase (PI3K) (–50±3%, n=69), aminogenistein (AMI) (10 mM), an antagonist of p56 tyrosine kinase (p56^Ick) (–35±4%, n=56), dioctanoyl glycerol (DOG, 1 mM), a stimulator of PKC (–30±9%, n=28) and KT5720 (1 mM), an antagonist of PKA (–20±8%, n=27) reduced ASP⁺-uptake. ASP⁺-kinetic measurements and k_i determination for cimetidine revealed effects on transporter trafficking as explanation for the inhibition of ASP⁺-uptake by these substances. Angiotensin II (AII, 1 nM) stimulated ASP⁺-uptake via Ca²⁺/calmodulin (40±7%, n=139), which assumedly arised from altered substrate affinity. Bafilomycin (BAF, 0.2 mM), an inhibitor of the vacuolar H⁺-ATPase, reduced ASP⁺-uptake (–52±3%, n=86). BAF did not prevent the AII effect, but abolished that of WOR. BAF seems to diminish the recycling rate of OC transporters (OCTs) and hence reduces the amount of transporters in the membrane. AII increases the substrate affinity of the OCTs. The involvement of the cytoskeleton in acute regulation of OCTs became obvious as the colchicine (COL) induced inhibition of microtubule polymerisation led to reduced ASP⁺-uptake (–60±7%, n=22). In immunhistochemical analyses WOR, AII and BAF demonstrated changes in membrane expression of mOCT2 in line with the findings described above.Supported by the Deutsche Forschungsgemeinschaft (DFG CI 107/4-1 to 2).