Lung fibrosis is characterized by increased deposition of extracellular matrix, especially collagens, and enhanced proliferation of fibroblasts. L-arginine is a key precursor of nitric oxide, asymmetrical dimethylarginine, and proline, an amino acid enriched in collagen. We hypothesized that L-arginine metabolism is altered in pulmonary fibrosis, ultimately affecting collagen synthesis. Expression analysis of key enzymes in the arginine pathway, protein arginine methyltransferases (Prmt), arginine transporters, and arginases, by quantitative (q) RT-PCR and Western blot revealed significant upregulation of arginase-1 and -2, but not Prmt or arginine transporters, during bleomycin-induced pulmonary fibrosis in mice. HPLC revealed a concomitant time-dependent decrease in pulmonary L-arginine levels. Arginase-1 and -2 mRNA and protein expression was increased in primary fibroblasts isolated from bleomycin-treated mice, as compared with controls, assessed by qRT-PCR and Western blot analysis. TGF-1, a key profibrotic mediator, induced arginase-1 and -2 mRNA expression in primary and NIH-3T3 fibroblasts. Treatment of fibroblasts with the arginase inhibitor, N^G-hydroxy-L-arginine (NOHA), attenuated TGF-1-stimulated collagen deposition, but not collagen mRNA expression or Smad signalling, in fibroblasts. In human lungs derived from patients with IPF, arginase activity was unchanged, but arginase-1 expression significantly decreased when compared with donor lungs. Our results thus demonstrate that arginase-1 is expressed and functionally important for collagen deposition in lung fibroblasts. TGF-1-induced upregulation of arginase-1 suggests an interplay between profibrotic agents and L-arginine metabolism during the course of lung fibrosis in the mouse, while species-specific regulatory mechanisms may account for the differences observed in mouse and man.