Imexon is a new antitumor agent with high activity in multiple myeloma. This drug induces apoptosis, oxidative stress and mitochondrial alterations. However, it was unknown whether imexon activates an intrinsic apoptotic pathway that is associated with activation of caspase-9 or an extrinsic pathway that is induced by receptor-mediated signals such as Fas ligand characterized by caspase-8 activation. In addition, we wanted to investigate the effect of imexon on Bcl-2 family proteins. In RPMI8226 myeloma cells, imexon activated caspase-9 and -3 in a time- and concentration-dependent manner. In contrast, cleavage of procaspase-8 was observed late and only after exposure to very high concentrations of imexon. Confocal microscopy confirmed that caspase-3 is also activated after treatment with imexon. High imexon concentrations activated caspase-3 and -9 at 12 h, while caspase-8 activation occurred only at 48 h. Imexon cytotoxicity was unchanged in three RPMI8226 cell lines with different levels (low, medium and high) of FAS expression. Similarly, the levels of Bcl-2, Bax and Bcl-xLwere unchanged in imexon-treated cells. However, Bcl-xLwas translocated to the mitochondria. These data suggest that imexon-induced oxidation activates the intrinsic or mitochondrial pathway of apoptosis, involving cytochromecrelease and activation of caspase-9 and -3.