Isolated rat hepatocytes were examined by EPR spectroscopy after exposure to inflammatory stimuli (interferon-γ [IFN-γ], tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and lipopolysaccharide [LPS]) in vitro, after in vivo immune activation by Corynebacterium parvum, and after exposure to · N0 and to nitroprusside (nitroferricyanide), an NO-donating nitrovasodilator. Hepatocytes exposed to IFN-γ, TNF-α, IL-1β, and LPS demonstrated the appearance of a g = 2.04 axial EPR signal indicative of the formation of nonheme iron-nitrosyl complexes. Concurrent incubation with L-NG-monomethylarginine (L-NMMA), a competitive inhibitor of · NO synthase, prevented the appearance of the signal. The g 2.04 signal was localized in the cytosolic fraction of hepatocyte extracts. Hepatocytes freshly isolated from C. parvum-treated rats exhibited a modest g = 2.04 signal, which was increased by a factor of approximately 2.5-fold upon subsequent 24-h culture in media without additional stimuli. This increase was prevented by L-NMMA in the culture medium and also by the presence of rat erythrocytes added to the culture. In the presence of erythrocytes, virtually all of the · NO produced was oxidized by reaction with intracellular hemoglobin within the erythrocyte, as judged by the relative amounts of nitrite and nitrate detected. These results suggest that in this model system · NO is sufficiently stable and diffusible to escape from the hepatocyte and diffuse into the erythrocyte without first reacting with oxygen or with intracellular iron at the site of its formation within the hepatocyte. Treatment of hepatocytes with exogenous · NO or nitroprusside generated an identical g = 2.04 signal of much greater intensity than with cytokines plus LPS. Treatment with nitroprusside also caused the appearance of a signal from pentacyanonitrosylferrate ion, verifying the previously reported metabolism of this nitrovasodilator by reduction and liberation of cyanide ion and · NO. These results indicate significant differences in intracellular nonheme iron nitrosylation in hepatocytes compared to cytotoxic activated macrophages, which may correlate with the differences in physiological function of · NO in these two systems.