A protein kinase C (PKC) activating factor (AF) has been identified in the extracellular medium of V3.17 vincristine resistant murine erythroleukemia (MEL) cells clone. The factor is a protein that stimulates the activity of PKC alpha and beta isozymes isolated from MEL cells, rat and mouse brain approximately 2 to 2.5 fold over the Vmax, respectively. AF promotes an identical activation in the presence of all the effectors but also when the amount of Ca2+ is reduced to microM concentration and in the absence of diacylglycerol (DAG). The factor shows a greater activating efficiency with PKC beta isozymes. AF binds to PKC presumably at the DAG binding site as suggested by the competition between phorbol dibutyrate and AF for binding to the kinase. Moreover, AF promotes the selective binding of PKC beta to natural or artificial membranes in the presence of microM concentrations of Ca2+. Altogether these results suggest the presence in MEL cells of a protein factor that can promote association of PKC to the membranes together with activation of the kinase, without the requirement for DAG formation. This could be visualized as a new mechanism for prolonged and selective activation of PKC.