Phosphorylation of Bcl2 at serine 70 may result from activation of a classic protein kinase C (PKC) isoform and is required for functional suppression of apoptosis by Bcl2 in murine growth factor-dependent cell lines (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). Human pre-B REH cells express high levels of Bcl2 yet remain sensitive to the chemotherapeutic agents etoposide, cytosine arabinoside, and Adriamycin. In contrast, myeloid leukemia-derived HL60 cells express less than half the level of Bcl-2 but are >10-fold more resistant to apoptosis induced by these drugs. The mechanism responsible for this apparent dichotomy appears to involve a deficiency of mitochondrial PKCalpha since 1) HL60 but not REH cells contain highly phosphorylated Bcl2; 2) PKCalpha is the only classical isoform co-localized with Bcl2 in HL60 but not REH mitochondrial membranes; 3) the natural product and potent PKC activator bryostatin-1 induces mitochondrial localization of PKCalpha in association with Bcl2 phosphorylation and increased REH cell resistance to drug-induced apoptosis; 4) PKCalpha can directly phosphorylate wild-type but not phosphorylation-negative and loss of function S70A Bcl2 in vitro; 5) stable, forced expression of exogenous PKCalpha induces mitochondrial localization of PKCalpha, increased Bcl2 phosphorylation and a >10-fold increase in resistance to drug-induced cell death; and () PKCalpha-transduced cells remain highly sensitive to staurosporine, a potent PKC inhibitor. Furthermore, treatment of the PKCalpha transformants with bryostatin-1 leads to even higher levels of mitochondrial PKCalpha, Bcl2 phosphorylation, and REH cell survival following chemotherapy. While these findings strongly support a role for PKCalpha as a functional Bcl2 kinase that can enhance cell resistance to antileukemic chemotherapy, they do not exclude the possibility that another Bcl2 kinase(s) may also exist. Collectively, these findings identify a functional role for PKCalpha in Bcl2 phosphorylation and in resistance to chemotherapy and suggest a novel target for antileukemic strategies.