Activation of protein kinase C (PKC) downregulates the human cationic amino acid transporters hCAT-1 (SLC7A1) and hCAT-3 (SLC7A3) (Rotmann, Strand, Martine and Closs, 2004 J. Biol. Chem. 279: 54185-92; Rotmann et al., 2006 Biochem. J 395: 117-23). However, others found that PKC increased arginine transport in various mammalian cell types, suggesting the expression of different arginine transporters might be responsible for the opposite PKC effects. We thus investigated the consequence of PKC activation by phorbol-12-myristate-13-acetate (PMA) in various human cell lines expressing leucine-insensitive system y⁺ (hCAT-1, hCAT-2B (SLC7A2) or hCAT-3) as well as leucine-sensitive system y⁺L (y⁺LAT1 (SLC7A7) or y⁺LAT2 (SLC7A6)) arginine transporters. PMA reduced system y⁺ activity in all cell lines tested independent of the hCAT isoform expressed, while mRNAs encoding the individual hCAT isoforms were either unchanged or increased. System y⁺L activity was also inhibited by PMA. The extent and onset of inhibition varied between cell lines, however, a PMA-induced increase in arginine transport was never observed. In addition, when expressed in Xenopus laevis oocytes, y⁺LAT1 and y⁺LAT2 activity was reduced by PMA and this inhibition could be prevented by the PKC inhibitor BIM I. In ECV304 cells, PMA-induced inhibition of systems y⁺ and y⁺L could be prevented by Go6976, a specific inhibitor of conventional PKCs. Thymelea toxin that activates preferentially classical PKC had a similar inhibitory effect as PMA. In contrast phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl, an activator of atypical PKC had no effect. These data demonstrate that both, system y⁺ and y⁺L are downregulated by classical PKC.