N-Benzyladriamycin-14-valerate (AD 198) is one of several novel anthracycline protein kinase C (PKC)-activating agents developed in our laboratories that demonstrates cytotoxic superiority over doxorubicin (Adriamycin; DOX) through its circumvention of multiple mechanisms of drug resistance. This characteristic is attributed at least partly to the principal cellular action of AD 198: PKC activation through binding to the C1b (diacylglycerol binding) regulatory domain. A significant dose-limiting effect of DOX is chronic, dose-dependent, and often irreversible cardiotoxicity ascribed to the generation of reactive oxygen species (ROS) from the semiquinone ring structure of DOX. Despite the incorporation of the same ring structure in AD 198, we hypothesized that AD 198 might also be cardioprotective through its ability to activate PKC-epsilon, a key component of protective ischemic preconditioning in cardiomyocytes. Chronic administration of fractional LD(50) doses of DOX and AD 198 to mice results in histological evidence of dose-dependent ventricular damage by DOX but is largely absent from AD 198-treated mice. The absence of significant cardiotoxicity with AD 198 occurs despite the equal ability of DOX and AD 198 to generate ROS in primary mouse cardiomyocytes. Excised rodent hearts perfused with AD 198 prior to hypoxia induced by vascular occlusion are protected from functional impairment to an extent comparable to preconditioning ischemia. AD 198-mediated cardioprotection correlates with increased PKC-epsilon activation and is inhibited in hearts from PKC-epsilon knockout mice. These results suggest that, despite ROS production, the net cardiac effect of AD 198 is protection through activation of PKC-epsilon.