Caspase activation is a central event in the execution phase of apoptosis and is associated with phosphatidylserine (PS) externalization and DNA fragmentation. We investigated the role of caspase activity in anticancer drug-induced PS externalization and DNA fragmentation in MTLn3 cells. Caspase activation (DEVD-AMC cleavage) occurred in a time- and concentration-dependent manner after exposure to doxorubicin, in association with cleavage of poly(ADP) ribose polymerase and protein kinase C delta, two caspase-3 substrates. Caspase activation was closely followed by oligonucleosomal DNA fragmentation and PS externalization as determined by flow cytometric analysis. Similar observations were made for etoposide and cisplatin. Inhibition of caspases with zVAD-fmk inhibited almost completely doxorubicin-induced DNA fragmentation as well as proteolysis of protein kinase C delta. In contrast, PS externalization induced by doxorubicin was only partly affected by caspase inhibition. Flow cytometric cell sorting demonstrated that DNA fragmentation in the remaining PS positive cells after doxorubicin treatment in the presence of zVAD-fmk was fully blocked. In conclusion, these data indicate that while DNA fragmentation in anticancer drug-induced apoptosis of MTLn3 cells is fully dependent on caspase activity, PS externalization is controlled by both caspase-dependent and caspase-independent pathways.