Evidence supporting increased nitric oxide (NO) in asthma is substantial, although the cellular and molecular mechanisms leading to increased NO are not known. Here, we provide a clear picture of the events regulating NO synthesis in the human asthmatic airway in vivo. We show that human airway epithelium has abundant expression of NO synthase II (NOSII) due to continuous transcriptional activation of the gene in vivo. Individuals with asthma have higher than normal NO concentrations and increased NOSII mRNA and protein due to transcriptional regulation through activation of Stat1. NOSII mRNA expression decreases in asthmatics receiving inhaled corticosteroid, treatment effective in reducing inflammation in asthmatic airways. In addition to transcriptional mechanisms, post-translational events contribute to increased NO synthesis. Specifically, high output production of NO is fueled by a previously unsuspected increase in the NOS substrate, l-arginine, in airway epithelial cells of asthmatic individuals. Finally, nitration of proteins in airway epithelium provide evidence of functional consequences of increased NO. In conclusion, these studies define multiple mechanisms that function coordinately to support high level NO synthesis in the asthmatic airway. These findings represent a crucial cornerstone for future therapeutic strategies aimed at regulating NO synthesis in asthma.