We have recently reported that the cytokine interferon-alpha (IFNα), commonly used in the treatment of cancer, induced a caspase-dependent apoptosis in tumor cell lines. The signaling mechanisms involved have not been defined. Here, we show that both proapoptotic Bcl-2 family members Bak and Bax were activated by IFNα, strictly in correlation with the induction of apoptosis. Using double stainings, we demonstrated that Bak was activated prior to cytochrome c (cyt c) release and caspase-3 activation, whereas activated Bax was only found in cells with released cyt c, mitochondrial depolarization, as well as activated caspase-3. Furthermore, IFNα-induced activation of Bak, and to a large extent also of Bax, was dependent on caspase activity. With the use of a panel of specific caspase inhibitors we found, however, that none of caspases-1 to-10 were responsible for this activation. Neither was the Ca 2+-dependent protease calpain nor the stress-activated p38 SAPK pathway significantly involved. Overexpression of Bcl-2 blocked apoptosis induced by IFNα totally abolished Bak activation, as well as decreased the amount of activated Bax. We conclude that IFNα induces Bak and Bax activation via distinct mechanisms involving an unknown protease, and that their activation is regulated by Bcl-2.