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This study characterizes the complex acute regulation of organic cation transporters (OCT) expressed in the basolateral membrane of isolated mouse proximal tubules (PT). A profound understanding of OCT transport regulation in mouse kidney is required especially as transgenic mouse models play a preeminent role in several pharmacological and physiological studies and in translational medicine and these transporters are involved in renal excretion of many drugs and organic substances used in experimental settings. The main OCT subtype in mouse PT is OCT1, and to a lesser extent (30%) OCT2, while OCT3 is only weakly expressed (1%).The fluorescent substrate ASP, 4-(-4-(dimethylamino) styryl-N-methylpyridinium, was used to quantify OCT transport across the basolateral membrane of isolated mouse proximal tubules (S2 and S3 segments) using a microtiter plate based fluorescence reader (excitation at 450 nm, emission at 590 nm). Tubules were mechanically isolated and transferred into a well of a 384 well microtiter plate. In PT (S2/S3 segments) of wildtype mice (WT) inhibition of phosphatidylinositol-3-kinase(PI3K)bywortmannin, of p56 tyrosine kinase (p56lck) by aminogenistein, stimulation of PKC by dioctanoyl glycerol and inhibition of PKA by KT5720 reduced ASP-uptake. Angiotensin II increased ASP-uptake via stimulation of Ca2+/calmodulin (CaM). In PT isolated from OCT2-defi-cient mice, which express mostly OCT1, the regulation pattern was identical. In PT from OCT1/2 double knockout mice, expressing only OCT3, ASP uptake was grossly reduced and only the p56lck inhibition altered transport. In HEK293 cells, stably expressing OCT1,2 or 3 identical findings were observed as in wildtype or OCT2-ko mice. In cells expressing OCT2 only inhibition of CaM activated transport while in cells expressing OCT3 again only the p56lck pathway altered transport. Analysing ASP-kinetics in PT of WT mice revealed that the PI3K, the p56lck, PKC or PKA pathways altered Vmax of the transport, suggesting a modification of transportertrafficking, while activation of CaM resulted in a decrease in Km suggesting an increase in substrate affinity. In conclusion, acute regulation of mouse OCT in proximal tubules involves similar regulation of the main subtypes OCT1 and OCT2 by various pathways, while the OCT3, which shows only very low expression, is only regulated by p56lck. These effects mostly involve transportertrafficking and only in the case of CaM a modification of substrate affinity. This is the first report unveiling the complex pattern of short-term regulation of OC-transport in freshly isolated mouse PTs. [ABSTRACT FROM AUTHOR]