Nuclear factor kappa-B (NF-B) is a versatile transcription factor that regulates a wide array of processes, including inflammation and survival, and plays a critical role in the etiology of inflammatory lung diseases. Nitric oxide (NO) has been suggested to play an anti-inflammatory role through S-nitrosation of components of NF-B pathway. NO production can be modulated by changing the availability of its substrate, L-arginine. Arginases compete with nitric oxide synthases (NOS) for their common substrate, L-arginine, and thereby have the potential to alter the signaling function of NO. The goal of the present study was to determine the impact of arginase manipulation on NO, and subsequent effects on NF-B activation, in lung epithelial cells. Our results demonstrate that reduction of arginase activity enhanced cellular content of NO, and S-nitrosated proteins (SNO), and resulted in decreases in tumor necrosis factor- (TNF or lipopolysaccharide (LPS)- stimulated NF-B DNA binding and transcriptional activity, in association with enhanced S-nitrosation of p50. The effects of arginase inhibition on NF-B were reversed by the generic NOS inhibitor, N--nitro-L-arginine methyl ester (L-NAME¹), suggesting a causal role for NO in the attenuation of NF-B induced by arginase suppression. Conversely, overexpression of arginase I decreased cellular SNO content and, enhanced IKK activity and NF-B DNA binding, and decreased S-nitrosation of p50. Collectively, our data point to a regulatory mechanism wherein NF-B is controlled through arginase-dependent regulation of NO levels, which may impact on chronic inflammatory diseases that are accompanied by NF-B activation and upregulation of arginases.