Protein kinase C (PKC) activators, such as the tumor-promoting phorbol esters, have been reported to protect several cell lines from apoptosis induced by a variety of agents. Recent evidence suggests that PKCepsilon is involved in protection of cardiac myocytes from hypoxia-induced cell death (Gray, M. O., Karliner, J. S., and Mochly-Rosen, D. (1997) J. Biol. Chem. 272, 30945-30951). We investigated the protective effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on U937 histiocytic lymphoma cells induced to undergo apoptosis by tumor necrosis factor-alpha (TNF-alpha) or by the specific PKC inhibitor calphostin C. U937 cells were transiently permeabilized with a peptide (epsilonV1-2) derived from the V1 region of PKCepsilon that has been reported to specifically block translocation of PKCepsilon. The epsilonV1-2 peptide blocked the inhibitory effect of TPA on both TNF-alpha- and calphostin C-induced apoptosis. A scrambled version of epsilonV1-2 and a peptide reported to inhibit PKCbeta translocation (betaC2-4) had no effect on the ability of TPA to inhibit apoptosis. These results suggest that PKCepsilon is required for the protective effect of TPA in TNF-alpha- and calphostin C-induced apoptosis. Furthermore, calphostin C reduced membrane-associated PKCepsilon activity and immunoreactivity, suggesting that PKCepsilon may play an important role in leukemic cell survival.