Staurosporine has long been used in vitro as an initiator of apoptosis in many different cell types, but the mechanism involved remains poorly understood. In the present study, we have examined the apoptosis-inducing potential of staurosporine in cultured melanoma cell lines and dissected the staurosporine-induced apoptotic signaling pathway. We report that although staurosporine activated Bax and the mitochondrial caspase-dependent apoptotic pathway, it also induced apoptosis of melanoma by caspase-independent pathways. The caspase-dependent apoptotic pathway was activated relatively soon after exposure to staurosporine and was associated with release of cytochrome c and Smac/DIABLO from mitochondria and cleavage of poly(ADP-ribose) polymerase and inhibitor of caspase-activated DNase. This pathway was inhibitable by broad caspase inhibitors. A second apoptotic pathway that appeared to be involved in late apoptotic events was caspase independent in that inhibitors of caspases did not prevent the late onset of apoptosis. Overexpression of Bcl-2 inhibited the early onset of apoptosis but not the later, caspase-independent pathway. Apoptosis-inducing factor may be responsible for the late apoptotic execution in that its translocation from mitochondria into the nucleus coincided with the late onset of apoptosis and could not be inhibited by either a pan-caspase inhibitor or overexpression of Bcl-2. Our results indicate that staurosporine is able to bypass resistance of melanoma cells to mitochondrial caspase-dependent apoptotic pathways; hence, derivatives of staurosporine may warrant further evaluation either alone or with other apoptosis-inducing agents.