Interferon alpha (IFNα) has been used in the treatment of several types of cancer for almost 30 years, yet the mechanism (s) responsible for its anti-tumoral action remains unknown. A variety of cellular responses, including inhibition of cell growth and induction of apoptosis are induced by IFNs, and apoptotic induction by this cytokine has been proposed to be of importance for both its anti-tumoral in addition to its anti-viral responses. The aim of the present study was to delineate the pathways activated during IFNα-induced apoptosis in malignant cell lines. We found that apoptosis induced by IFNα was associated with activation of caspases-1,-2,-3,-8 and-9 and that this activation was a critical event. Caspase-3 activation was dependent on activity of caspases-8 and-9, moreover, activation of caspase-8 seems to be the upstream event in IFNα-induced caspase cascade. We also found loss of mitochondrial membrane potential as well as release of cytochrome c post IFN-treatment, clearly implicating the involvement of mitochondria in IFN-mediated apoptosis. Furthermore, IFNα-induced apoptosis was found to be independent on interactions between the Fas-receptor and its ligand. These studies form the basis for further investigations aiming to improve IFN therapy and the development of future strategies to overcome the IFN resistance observed in some malignancies.