The ability of the protein kinase C down-regulator bryostatin 1 to potentiate 1-beta-D-arabinofuranosylcytosine (ara-C)-induced apoptosis was examined in human leukemia cells (U937) over-expressing the antiapoptotic protein Bcl-x(L). Coadministration of bryostatin 1 with ara-C resulted in enhanced cytosolic release of cytochrome c and Smac/DIABLO, procaspase-3 and -9 activation, loss of mitochondrial membrane potential (Deltapsi(m)), poly(ADP-ribosyl)phosphorylase degradation, apoptosis, and loss of clonogenic survival in U937/Bcl-x(L) cells, although effects were not as marked as in empty-vector control cells. Whereas the broad caspase inhibitor ZVAD-fluoromethyl ketone blocked ara-C/bryostatin 1-mediated caspase activation, loss of Deltapsi(m, )and apoptosis in U937 cells, it failed to diminish cytochrome c release. In contrast, ectopic expression of Bcl-x(L) blocked cytochrome c redistribution as well as all other events involved in ara-C/bryostatin 1-mediated apoptosis. The ability of ectopic expression of cytokine response modifier A to attenuate, albeit partially, bryostatin 1-mediated potentiation of ara-C-related apoptosis suggested a contributory role for activation of the extrinsic pathway in this phenomenon. Finally, the F(0)F(1) ATPase inhibitor oligomycin effectively blocked cytochrome c release as well as loss of Deltapsi(m) and apoptosis in U937/Bcl-x(L) cells. Together, these findings support the concept that bryostatin 1 potentiates ara-C lethality in human leukemia cells ectopically expressing Bcl-x(L) by diminishing the capacity of this antiapoptotic protein to antagonize cytochrome c release. In addition, they raise the possibility that activation of caspase cascades operating independently of Bcl-x(L)-associated mitochondrial actions may also contribute to enhanced lethality.