Previous studies from our laboratory have shown that the MDCK cell taurine transporter (pNCT) is down-regulated by protein kinase C (PKC) activation. The fourth intracellular segment (S4) of pNCT appears to participate directly in taurine transporter inactivation that is modulated by PKC phosphorylation. Six putative PKC phosphorylation sites are located in pNCT including Ser-45, Thr-175, Ser-215, Thr-242, Ser-322, and Thr-581. These PKC consensus sites are 100% conserved in all the taurine transporters that have been cloned from different animal species. We have individually mutated each of these residues in pNCT by site-directed mutagenesis and compared the function of the mutants with that of wild type transporter by expressing the transporters in Xenopus laevis oocytes. In this study we found that Ser-322, located in S4, is the critical site of PKC phosphorylation. When Ser-322 of pNCT was replaced by alanine (S322A) and expressed in oocytes, taurine transport activity was increased 3-fold as compared with wild-type pNCT (87.0±13.7 vs 28.3±6.0 pmol/h/oocyte). Activation of PKC by the active phorbol ester PMA (phorbol 12-myristate 13-acetate) did not affect taurine transport by mutant S322A expressed in oocytes. Kinetic analysis showed that the mutation mainly affected the Vmax (170.0 vs 66.9 pmol/h/oocyte, mut vs wt) rather than the Km of the taurine transporter(10.5 vs 7.3 uM, mut vs wt). Mutation of other PKC concensus sites did not affect taurine transporter activity expressed in oocytes. We conclude that only Ser-322 is critical in PKC down-regulation of taurine transporter activity, and that regulation may occur largely by changes in the numbers of active transporters in the plasma membrane into inactive transporters by PKC phosphorylation.