Both arginase isoforms (AI and AII) regulate high-level NO production by the inducible NOS, but whether the arginase isoforms also regulate low-level NO production by neuronal NOS (nNOS) is not known. In this study, 293 cells that stably overexpress nNOS gene (293nNOS cells) were transfected with rat AI (pEGFP-AI) or AII (pcDNA-AII) plasmids, and nitrite production was measured with or without supplemental L-arginine. Transfection with pEGFP-AI increased AI expression and activity 10-fold and decreased intracellular l-arginine by 50%. Nitrite production was inhibited by >80% when no l-arginine was supplemented but not when 1 mM L-arginine was present. The inhibition was reversed by an arginase inhibitor, N(omega)-hydroxy-L-arginine. Transfection with pcDNA-AII increased AII expression and activity but had little effect on nitrite production even if no l-arginine was added. These results suggest that, in 293nNOS cells, AI was more effective in regulating NO production by nNOS, most likely by competing for L-arginine.