Indirect evidence has suggested that P-glycoprotein (P-gp), the multidrug transporter, is phosphorylated by protein kinase C (PKC) and that phosphorylation modulates its transport function. To address the first premise more directly, ie., that P-gp is phosphorylated by PKC, we investigated the interaction between P-gp and PKC in sensitive and multidrug resistant MCF-7 and KB human carcinoma cell lines. We found that P-gp and PKC were coimmunoprecipitated from the multidrug-resistant cell lines MCF-7/AdrR and KB-V-1, using antibodies to either protein. The association between the two proteins was enhanced by phorbol 12-myristate 13-acetate, an analogue of diacylglycerol that induces translocation of PKC to the plasma membrane. The anti-P-gp immunoprecipitates contained PKC activity as measured by direct phosphorylation reactions. The interaction of PKC with P-gp displayed isozyme specificity: PKC-alpha, -beta, gamma, -epsilon, and -phi, but not -delta, -mu, -zeta, -lambda, were found to coimmunoprecipitate with P-gp. These studies indicate that P-gp closely interacts with PKC and serves as a substrate, and that specific isozymes of this kinase may be involved in the phosphorylation of the multidrug transporter.