The organic cation transporter (OCT3) is widely expressed in various organs, such as kidney, heart and brain. OCT3 is involved in the disposition of many exogenous and endogenous compounds and is emerging as a regulator of brain serotonin concentration possibly playing a role in mood-related behaviors. Aim of this work was to study the transport properties of mouse OCT3 (mOCT3) stably expressed in human embryonic kidney cells and to compare its acute regulation in expression system with that in freshly isolated proximal tubules from OCT1 and 2 double knockout mice, where OCT3 is the only OCT expressed. As mOCT3 substrate the fluorescent organic cation 4-(4-(dimethyl-amino)styril)-methylpyridinium iodide (ASP⁺) was used. ASP⁺-uptake was pH- and Na⁺-independent and membrane potential-dependent. Transport of ASP⁺ (K_m=2.5mM) was inhibited by corticosterone, tetrapentylammonium, methylphenylpyridinium and cimetidine with IC50 of 4, 73, 108 and 139mM, respectively. Serotonin was substrate of mOCT3. Both serotonin and the selective serotonin reuptake inhibitor Fluoxetine concentration dependently inhibited ASP⁺-uptake by mOCT3. Inhibition of p56^lck tyrosine kinase (5mM Aminogenistein, 10 min) significantly reduced ASP⁺-uptake in expression system (–16±2, n=83) and in mouse tubules (–29±6%, n=38), while activation of PKA (1mM Forskolin), PKC (1mM DOG), inhibition of PI3K (0.1mM Wortmannin), and Ca²⁺/Calmodulin (5mM Calmidazolium) remained without effect in both systems. In conclusion, the transport via mOCT3 obeys known general properties of OCT and seems to be involved in serotonin uptake modulation. Moreover, mOCT3 is endogenously regulated in vitro and ex vivo by p56^lck tyrosine kinase. Supported by the Deutsche Forschungsgemeinschaft (DFG CI 107/4-1 to 2).