We previously reported that topical application of 12-o-tetradecanoylphorbol-13-acetate to mouse skin causes phosphorylation of epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34). In the accompanying paper, p40 was identified as creatine phosphokinase B. Here we report that both in intact cells and in a cell-free system, phosphorylation of creatine hosphokinase B by protein kinase C resulted in an increase in its ability to catalyze the transfer of the high-energy phosphate of phosphocreatine to ADP, thereby producing ATP. H-7, a specific inhibitor of protein kinase C was found to abolish the increase in enzyme activity. Lineweaver-Burk plot analysis indicated that the increased activity was mostly due to a decreased Km for phosphocreatine. Phosphorylation and activation of creatine phosphokinase B may be a physiological response to maintain ATP balance when a protein kinase C pathway is stimulated.