A variety of approaches have been employed to demonstrate that the interaction of protein kinase C beta II with phorbol ester-containing membranes is reversible, is not accompanied by significant insertion of the protein into the hydrophobic core of the membrane, and is qualitatively similar to the interaction with diacylglycerol (DG). First, we show that under conditions when protein kinase C is bound with equal affinity to membranes containing either DG or phorbol myristate acetate (PMA), increasing ionic strength causes a similar reduction in membrane binding. The similar sensitivity to ionic strength indicates that the forces mediating the binding of protein kinase C to PMA are not significantly different from those mediating the binding to DG. At sufficiently high concentrations of PMA and relatively low concentrations of phosphatidylserine, the binding of protein kinase C to membranes became markedly less sensitive to ionic strength, suggesting that under these conditions direct non-electrostatic interactions with PMA dominate over electrostatic interactions with the lipid headgroups. Importantly, regardless of the strength of the interaction with PMA, protein kinase C exchanges between vesicle surfaces: protein kinase C bound first to phorbol ester-containing multilamellar vesicles exchanged to large unilamellar vesicles upon addition of an excess surface area of the latter. Lastly, the enzyme's intrinsic tryptophan fluorescence was not quenched by bromines located at various positions in the hydrophobic core of the membrane. In contrast, the enzyme's tryptophan fluorescence was significantly quenched by probes positioned at the membrane surface. In summary, our results are consistent with protein kinase C binding reversibly to PMA- or DG-containing membranes primarily via interactions at the membrane interface.