Procytokine processing by caspase-1 is required for the maturation and release of IL-1β and IFN-γ-inducing factor (IGIF)(or IL-18) from activated macrophages (Mφ). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1β-converting enzyme (ICE) or caspase-1 activity. Activated RAW264. 7 cells released four to five times more IL-1β, but not TNF-α, in the presence of the NO synthase inhibitor N G-monomethyl-l-arginine. Stimulated peritoneal Mφ from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1β if exposed to N G-monomethyl-l-arginine, whereas Mφ from iNOS knockout mice (iNOS−/−) did not. Inhibition of NO synthesis in stimulated RAW264. 7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-dl-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1β and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1β and IFN-γ than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1β and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1β and IGIF release.