L-Arginine metabolism by the arginase (ARG) and nitric oxide synthase (NOS) families of enzymes is important in NO production, and imbalances between these pathways contribute to airways hyperresponsiveness (AHR) in asthma. To investigate the role of arginase isozymes in AHR we determined the protein expression of ARG1 and ARG2, the NOS isozymes, as well as other proteins involved in L-arginine metabolism, in lung tissues from human asthmatics and in acute (3-week) and chronic (12-week) murine models of ovalbumin (OVA)-induced airways inflammation. Expression of ARG1 was increased in human asthma, while ARG2, NOS isoforms, and the other L-arginine-related proteins (i.e., CAT1, CAT2, agmatinase, and ornithine decarboxylase) were unchanged. In the acute murine model of allergic airways inflammation, augmentation of ARG1 expression was similarly the most dramatic change in protein expression. However, ARG2, NOS1, NOS2 and agmatinase were also increased, while NOS3 expression was decreased. Arginase inhibition in vivo, with nebulized S-(2-boronoethyl)-L-cysteine (BEC), attenuated the methacholine responsiveness of the central airways in mice from the acute model. Further investigations in the chronic murine model revealed an expression profile that more closely paralleled the human asthma samples; i.e., only ARG1 expression was significantly increased. Interestingly, in the chronic mouse model, which generates a remodeling phenotype, arginase inhibition attenuated methacholine responsiveness of both the central and peripheral airways. The similarity in arginase expression between human asthma and the chronic model, and attenuation of AHR following in vivo treatment with an arginase inhibitor suggests the potential for therapeutic modification of arginase activity in asthma.