The activation of type I (gamma), II (beta) and III (alpha) protein kinase C (PKC) subspecies by phosphatidylserine (PS) and diacylglycerol (DAG) is inhibited by micromolar concentrations of triphenylacrylonitrile (TPE) antiestrogens. TPE A (with p-hydroxy and p-diethylaminoethoxy groups on the 3- and 3'-phenyl rings, respectively) interacts with PS-vesicles as well as with the regulatory domain of PKC, probably at a site different from the Ca2+ and DAG binding sites. The interaction of TPE A with the regulatory domain of enzyme is very slow. Apparently, TPE A does not interact with the catalytic domain of PKC. In contrast, another TPE derivative, TPE B (with a p-hydroxy group on each of the three phenyl rings) inhibits the enzyme activity in a competitive manner with respect to ATP, suggesting that this TPE interacts with the catalytically active site of the enzyme. It seems likely that various TPE antiestrogen derivatives may exert their inhibitory action on PKC by multiple different mechanisms.