Recent studies have documented evidence for the death of smooth muscle cells (SMCs) within advanced human atheroma. These lesions contain macrophages and T lymphocytes in addition to SMCs. We therefore investigated whether interferon-γ (IFN-γ), a cytokine secreted by T lymphocytes, or interleukin-1β (IL-1β) and tumor necrosis factor–α (TNF-α), two cytokines characteristically produced by activated macrophages, can trigger apoptosis of vascular SMCs. Simultaneous treatment with IFN-γ and TNF-α and/or IL-1β but not with each cytokine alone promoted death of human and rat SMCs. Exposure for 48 hours to a combination of IFN-γ (400 U/mL), TNF-α (400 U/mL), and IL-1β (100 U/mL) significantly (P<.001) increased the accumulation of oligonucleosomes comprising DNA fragments and histones in human SMCs. Electrophoresis of genomic DNA showed internucleosomal fragments of genomic DNA isolated from the cytokine-cotreated SMCs of both humans and rats. These cells exhibited morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. In situ 3′ end labeling of DNA fragments with terminal transferase confirmed the fragmentation of genomic DNA in these cells. Simultaneous treatment with IFN-γ and TNF-α or IL-1β induced elaboration of nitrite, an end product of nitric oxide, in rat but not human SMCs. N^G-monomethyl-l-arginine inhibited nitrite accumulation and also partly blocked cytokine-induced apoptosis of rat SMCs but had little effect on human SMCs, suggesting operation of both nitric oxide–dependent and –independent mechanisms for cytokine-induced apoptosis in vascular SMCs. Production of immune cytokines by vascular cells and/or infiltrating leukocytes may regulate apoptotic death of SMCs during atherogenesis.