The role of antioxidants in reducing cancer initiation and progression has been highlighted in recent years. Not only antioxidants limit cancer cell growth but also, in some situations, they promote the effectiveness of conventional treatments. Melatonin, an endogenously synthesized antioxidant, reduces cell growth of several tumor types both in vivo and in vitro. Additionally, the indole limits the collateral damage induced by many chemotherapeutic agents. By using a cellular model of human prostate cancer, we studied the ability of melatonin to enhance apoptosis induced by tumor necrosis factor or gamma radiation. It has been reported that melatonin reduces prostate cancer cell growth and, more recently, it promotes cell differentiation. In this work, we also show that melatonin elevates p21 protein levels and increases antioxidant capacity of prostate cancer cells. In addition, melatonin significantly enhances hrTNFalpha induced cell death by decreasing NFkappaB activation. Bcl-2 and survivin down-regulation appears to be associated to apoptosis stimulation under NFkappaB inhibition. On the contrary, melatonin does not promote irradiation-induced cell death due to an increment in intracellular glutathione content. In conclusion, prevention of NFkappaB activation by melatonin enhances the effectiveness of cytokine treatment in prostate cancer cells but it is not sufficient to enhance cell death triggered by other therapies which generate free radicals. A crucial role of glutathione in survival mechanisms of prostate cancer cells should be carefully considered.