The tumor promoter receptor protein kinase C (PKC) has been implicated as a key enzyme in cellular growth regulation. It is, therefore, believed that specific PKC inhibitors may include effective antiproliferative agents. Previously, we have shown that the antiestrogen tamoxifen and related triphenylethylenes are potent inhibitors of PKC. Although the mechanism of inhibition of PKC by triphenylethylenes clearly involves nonspecific interactions between the antiestrogens and the lipid cofactor of PKC, we recently demonstrated that PKC itself has specific triphenylethylene-binding sites, suggesting that the inhibitory mechanism also involves specific drug-protein interactions. In this report, we characterize the direct interactions between PKC and triphenylethylenes and demonstrate their relevance to the inhibitory action of triphenylethylenes against PKC. We show (a) that the triphenylethylene-binding sites of PKC are located in the catalytic domain of the enzyme, (b) that MgATP (i.e., 10 mM MgCl2 plus 1 mM ATP) competes with the triphenylethylenes for binding sites on PKC, and (c) that triphenylethylenes are competitive inhibitors of PKC with respect to MgATP. Taken together, these data provide strong evidence that triphenylethylenes can inhibit PKC by binding directly to the ATP-binding region of the active site of the enzyme. The specific interactions between triphenylethylenes and PKC characterized here may provide a rationale for developing more specific PKC inhibitors.