Rationale: Increasing evidence suggests that lung mechanics and structure are maintained in part by an intimate balance between the L-arginine-metabolizing enzymes nitric oxide synthase (NOS) and arginase. Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of NOS. The role of ADMA in the regulation of NOS and arginase in the airways has not yet been explored. Objectives: To investigate the role of ADMA in lung physiology. Methods: A murine model of continuous subcutaneous ADMA infusion via osmotic minipump was utilized for assessment of elevated ADMA in vivo and primary lung fibroblasts were utilized for in vitro assessments. Two weeks following minipump placement, animals were anesthetized, mechanically ventilated and lung mechanical responses were evaluated. Lungs were assessed histologically and biochemically for collagen content, arginase activity, and arginase protein levels. Lung lavage fluid was assessed for cellularity, nitrite, urea and cytokine concentrations. Measurements and Main Results: ADMA infusion resulted in significantly enhanced lung resistance and decreased dynamic compliance in response to methacholine. These physiological changes were associated with significantly increased lung collagen content in the absence of inflammation. Significant decreases in lung fluid nitrite were accompanied by elevated lung fluid urea and arginase activity in lung homogenates. These changes were reversed in mice 4 weeks following completion of ADMA administration. In addition, treatment of primary mouse lung fibroblasts with ADMA stimulated arginase activity and collagen formation in vitro. Conclusions: These data support that ADMA may play a role in airway diseases including asthma and pulmonary fibrosis through NOS inhibition and enhancement of arginase activity.