All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly (ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-β and had been correlated with apoptosis induction. Because IFN-β induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-β for 16–24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone,> 30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-β and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly (ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-β and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-β and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.