Arginase, which exists as the isoforms arginase I and II, catalyzes the hydrolysis of arginine to ornithine and urea. Ornithine is the principal precursor for production of polyamines, which are required for cell proliferation. Rat aortic smooth muscle cells (RASMC) contain constitutive arginase I, and arginase inhibitors cause inhibition of cell proliferation. The objective of this study was to determine whether the elevated expression of arginase I in RASMC causes increased cell proliferation. RASMC were stably transfected with either rat arginase I cDNA or a β-galactosidase control expression plasmid. Western blots and arginase enzymatic assays revealed high-level expression of cytosolic arginase I in arginase I-transfected RASMC. Moreover, this observation was associated with the increased production of urea and polyamines and higher rates of RASMC proliferation. The two selective inhibitors of arginase, N^G-hydroxy-l-arginine and S-(2-boronoethyl)-l-cysteine, inhibited arginase and decreased the production of urea and polyamines in arginase I-transfected RASMC, all of which were associated with the inhibition of cell proliferation. This study demonstrates that elevated arginase I expression increases RASMC proliferation by mechanisms involving increased production of polyamines. These observations suggest that arginase I plays a potentially important role in controlling RASMC proliferation.