We investigated whether murine peritoneal macrophages treated with cisplatin or interferon (IFN)-γ alone, or in combination, could undergo apoptosis, and whether this results either from the cytotoxic effect of the activating agents or indirectly in an autocrine manner by the cytotoxic molecules released by them upon activation. Our data suggest that cisplatin, which has been shown to induce apoptosis in a number of normal as well as tumor cell types, did not induce apoptosis in murine peritoneal macrophages nor was apoptosis caused by IFN-γ. However, combined treatment with cisplatin and IFN-γ induced apoptosis in macrophages as studied by percent DNA fragmentation assay, qualitative analysis of DNA on agarose gel electrophoresis, and morphological and nuclear alterations studied by phase contrast and fluorescence microscopy. The factor responsible for inducing apoptosis in macrophages was found to be a higher concentration of NO produced by them upon activation with cisplatin and IFN-γ. Macrophages treated with cisplatin or IFN-γ alone produced a low level of NO and did not undergo apoptosis. The inhibitor of NO synthase, L-NMMA, prevented apoptosis in macrophages treated with cisplatin and IFN-γ, suggesting the involvement of NO in the induction of apoptosis in macrophages. The role of NO in inducing apoptosis in macrophages was further confirmed by the observation that direct treatment with sodium nitroprusside, a NO donor, resulted in apoptosis in macrophages. We have also shown that NO-induced apoptosis in macrophages activated with cisplatin and IFN-γ requires activation of an endonuclease, as the endonuclease inhibitor, aurine tricarboxylic acid, prevented apoptosis in them.