On the basis of in vitro inhibition of tumor cell growth, IFNs have been generally considered to be antiproliferative proteins. To probe further the potential mechanisms of the antitumor effects of IFNs, we have assessed apoptosis in response to IFN-alpha2 and IFN-beta in cell lines of varied histologies, with a focus on melanomas. Many of the cell lines tested underwent apoptosis in response to IFN-beta, as assessed both by Annexin V and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. In general, IFN-beta had greater growth inhibitory and proapoptotic effects than IFN-alpha2 on all cell lines. The melanoma cell line WM9, sensitive to growth inhibition by IFNs, had a greater degree of apoptosis than A375 melanoma cells, which were largely resistant to antigrowth effects of IFNs. IFN-beta-induced apoptosis was dependent on activation of the caspase cascade with cleavage of caspases 3, 8, and 9 and of the caspase 3 substrate, poly(ADP-ribose) polymerase. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl keton or benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl keton, inhibited IFN-beta-induced apoptosis. Other changes associated with apoptosis, including the movement of cytochrome c from mitochondria to cytoplasm and DNA fragmentation, were also identified in response to IFN-beta. Apo2L ligand [tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)] was one of the early genes induced by IFN-beta in apoptosis-sensitive WM9 cells. Other sensitive melanoma cell lines had a similar IFN-beta-specific induction of TRAIL. Neutralizing antibody to TRAIL inhibited IFN-beta-induced apoptosis in WM9 cells. In resistant A375 cells, IFN-beta did not induce TRAIL/Apo2L expression. Thus, induction of TRAIL by IFNs in some tumor types may initiate the apoptotic cascade. This study offers another mechanism for the antitumor effects of IFNs.