We have studied the production of nitric oxide (NO) and superoxide by murine peritoneal macrophages during activation. The production of NO was induced by activation of cells with recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS). Phorbol 12-myristate 13-acetate (PMA)-induced formation of superoxide also increased during activation. However, NO released by the activated macrophages exerted the inhibitory effect on the superoxide formation in the same cells. This fact is supported by the increased production of superoxide when the cells were treated with NG-monomethyl-L-arginine (NGMMA) in addition to stimulation with rIFN-gamma and LPS. The production of superoxide was also inhibited by treatment with sodium nitroprusside (SPN), which spontaneously released nitric oxide in vitro, and at the same time there was increased adenosine diphosphate (ADP)-ribosylation of 37 kDa proteins of the cytoplasm. The 3-aminobenzamide (3-AB) treatment, which decreased ADP-ribosylation, partially reversed SNP-induced inhibition of superoxide generation in macrophages. The above data provide evidence that NO decreases superoxide formation possibly via ADP-ribosylation.