We have previously demonstrated that tamoxifen and related triphenylethylene compounds are potent inhibitors of protein kinase C (PKC). The present study demonstrates that PKC binds specifically and reversibly to the antiestrogen N-didesmethyltamoxifen when the drug is coupled to CNBr-activated agarose through its primary amine, in the absence of lipid and other cofactors of the enzyme. PKC did not bind to 4-hydroxytamoxifen, which had been immobilized on epoxy-activated Sepharose through its hydroxyl moiety. This shows that the binding of PKC to immobilized N-didesmethyltamoxifen was not merely due to hydrophobic interactions, since N-didesmethyltamoxifen and 4-hydroxytamoxifen have nearly identical hydrophobicities. These results demonstrate that PKC has specific triphenylethylene-binding sites, which may mediate the inhibition of PKC activity by these antiestrogens.