We have previously shown that the protein kinase C (PKC) signal transduction pathway regulates cell death by the DNA damaging agent cis-diamminedichloroplatinum (II)(cDDP). In the present study we have investigated how PKC influences the sequence of events that are triggered by cDDP-induced DNA damage. cDDP caused activation of caspases-8,-9,-3,-7 and cleavage of PKCδ. Rottlerin, a selective inhibitor of novel PKCδ, blocked activation of caspases, proteolytic activation of PKCδ and cell death induced by cDDP. In contrast, Gö 6976, an inhibitor of conventional PKCα and βI, did not prevent cDDP-induced caspase activation and cDDP cytotoxicity. In HeLa cells, PKCδ was distributed both in the cytosol and heavy membrane (HM) fraction containing mitochondria. While caspase-8 was primarily cytosolic, a small amount of caspases-9,-7 and-3 could be detected in the HM fraction. cDDP caused a time-dependent increase in Cytochrome c release from the mitochondria and processing of both cytosolic and membrane-associated caspases, as well as proteolytic cleavage of PKCδ. Rottlerin attenuated late but not early release of Cytochrome c by cDDP. It, however, inhibited activation of caspases and proteolytic cleavage of PKCδ in both cytosolic and HM fractions. The antiapoptotic effect of rottlerin was evident when it was added together with or following cDDP addition but not when added after cDDP was removed from the medium. Thus, the PKCδ inhibitor acts at an early stage of the cDDP-induced cell death pathway that precedes caspase activation. Cell Death and Differentiation (2001) 8, 899–908