To test the role of caspase 3 in apoptosis and in overall cell lethality caused by the protein kinase inhibitor staurosporine, we compared the responses of MCF-7c3 cells that express a stably transfected CASP-3 gene to parental MCF-7:WS8 cells transfected with vector alone and lacking procaspase-3 (MCF-7v). Cells were exposed to increasing doses (0.15–1 μM) of staurosporine for periods up to 19 h. Apoptosis was efficiently induced in MCF-7c3 cells, as demonstrated by cytochrome c release, processing of procaspase-3, procaspase-8, and Bid, increase in caspase-3-like DEVDase activity, cleavage of the enzyme poly(ADP-ribose) polymerase, DNA fragmentation, changes in nuclear morphology, and TUNEL assay and flow cytometry. For all of these measures except cytochrome c release, little or no activity was detected in MCF-7v cells, confirming that caspase-3 is essential for efficient induction of apoptosis by staurosporine, but not for mitochondrial steps that occur earlier in the pathway. MCF-7c3 cells were more sensitive to staurosporine than MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to killing by staurosporine when evaluated by a clonogenic assay. A similar distinction between apoptosis and loss of clonogenicity was observed for the cancer chemotherapeutic agent VP-16. These results support our previous conclusions with photodynamic therapy: (a) assessing overall reproductive death of cancer cells requires a proliferation-based assay, such as clonogenicity; and (b) the critical staurosporine-induced lethal event is independent of those mediated by caspase-3.