A significant increase in the induction of inducible nitric-oxide synthase (iNOS) protein expression and in the levels of nitrite plus nitrate was observed in rat aortic smooth muscle cells (RASMCs) stably transfected with catalase (RASMC-2C2) as compared with empty vector-transfected RASMC-V4 cells after exposure to cytokines and lipopolysaccharide. The increased expression of iNOS protein in the RASMC-2C2 cells was associated with a significant activation of nuclear transcription factor kappaB, one of the transcriptional regulators of iNOS expression. The induction of iNOS was also accompanied by increased protein tyrosine nitration in both cell types as revealed by immunocytochemical staining and high pressure liquid chromatography with on-line electrospray ionization tandem mass spectrometry. Nitrotyrosine formation was inhibited by 1400W, an iNOS inhibitor, by 4-(2-aminoethyl) benzenesulfonyl fluoride, an inhibitor of NADPH oxidase, and by the superoxide dismutase mimetic M40403, but not by the peroxidase inhibitor 4-aminobenzoic hydrazide. Electron microscopy using affinity-purified anti-nitrotyrosine antibodies revealed labeling at the cytosolic side of the rough endoplasmic reticulum membranes, in the nucleus, occasionally in mitochondria, and consistently within the fibrillar layer underneath the plasma membrane. Collectively, the data in this model system indicate that hydrogen peroxide, by inhibiting the activation of nuclear transcription factor kappaB, prevents iNOS expression, whereas superoxide contributes in a precise pattern of intracellular protein tyrosine nitration.