Peripheral blood mononuclear cells (PBMCs) obtained from patients with advanced melanoma but not from healthy individuals were found to undergo spontaneous ex vivo apoptosis upon incubation in medium. PBMCs were evaluated for evidence of apoptosis using Annexin V binding, caspase-3 activation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). PBMCs of patients with melanoma contained a significantly higher proportion(P = 0.0027) of spontaneously apoptotic cells than PBMCs of controls after 24-h incubation in medium alone. The relative proportion of activated Fas⁺ and tumor necrosis factor receptor 1-positive (TNFR1⁺) PBMCs was significantly higher in patients with melanoma than that observed in controls. To demonstrate that the TNF family of receptors and ligands was involved in this type of apo-ptosis, PBMCs were incubated in the presence of agonistic anti-Fas antibody (CH-11) or TNF-α. The proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive PBMCs undergoing spontaneous apoptosis was found to be comparable with that induced by CH-11 antibody or TNF-α. Three-color flow cytometry revealed that CD3⁺Fas⁺ T cells were especially sensitive to apoptosis and were preprogrammed in vivo to die. Apoptosis occurred in all subsets of PBMCs but was significantly higher(P = 0.01) in the CD3⁺ CD8⁺T-cell subset in patients relative to controls. In two patients with melanoma, who responded clinically to dendritic cell-based peptide vaccines, the proportion of apoptotic T cells was decreased by half after therapy. In patients who were treated previously with vaccination-based therapies, levels of T-cell apoptosis were lower than in the other melanoma patients. The observed accelerated death of T cells, which are activated and susceptible to apoptosis in patients with melanoma, may contribute to a rapid turnover of immune cells,resulting in a decreased immunocompetence.