Bcl-2 inhibits apoptosis induced by numerous antitumor drugs, including doxorubicin and daunorubicin and is, thus, a major impediment to successful cancer chemotherapy. Here, we report the ability of a novel family of nonnuclear targeted anthracyclines to induce rapid apoptosis in cells despite Bcl-2 or Bcl-X(L) expression. Typified by N-benzyladriamycin-14-valerate (AD 198) and N-benzyladriamycin-14-pivalate (AD 445), this family of compounds binds to the C1 regulatory domain of protein kinase C (PKC), competitively inhibits phorbol ester binding in cell-free studies, and induces PKC translocation in intact cells. PKC-delta has an established role as a pro-apoptotic protein through the association of the holoenzyme or catalytic fragment with mitochondria. In proliferating 32D.3 myeloid cells, or in 32D.3 cells engineered to overexpress Bcl-2, substantial levels of PKC-delta are associated with mitochondria. However, after a 1-h exposure to 5 microM AD 198, cytochrome c release, caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, PKC-delta cleavage, and DNA fragmentation are observed. Pretreatment of 32D.3 cells with the selective PKC-delta inhibitor, rottlerin, but not the general PKC inhibitor, GF 109203X, or PKC-alpha and -beta inhibitor, Gö 6976, delayed the 50% cell kill to >24 h for control and Bcl-2 overexpressing 32D.3 cells treated with 5 microM AD 198. Rottlerin delayed PKC-delta and PARP cleavage to >20 h post-drug exposure and also delayed mitochondrial membrane depolarization. In contrast, the pan-caspase inhibitor Z-Val-Ala-Asp-CH2F blocked PKC-delta and PARP cleavage, but not mitochondrial membrane depolarization. These results suggest that AD 198 induces mitochondrial-dependent apoptosis in 32D.3 cells by activating PKC-delta holoenzyme on mitochondria, which, in turn, overrides the antiapoptotic effects of Bcl-2.