In macrophages and many other cell types,l-arginine is used as a substrate by both nitric oxide synthase (NOS) and arginase to produce nitric oxide (NO) and urea, respectively. Because the availability ofl-arginine is a major determinant for NO synthesis in the activated macrophage, we hypothesized that NO production may be reduced by arginase via depleting the common substrate in this cell type. To test this hypothesis, we investigated the effect of an arginase inhibitor,l-norvaline, on NO production in J774A.1 mouse macrophages activated by lipopolysaccharide (LPS, 1.0 μg/ml) for 22 h. In the absence of LPS, macrophages produced a low level of NO. In contrast, NO production from these cells was significantly increased in the presence of LPS. Increasing extracellular levels ofl-arginine (0.01–0.8 mM) produced a concomitant increase in NO production of activated macrophages. l-Norvaline (10 mM), which specifically inhibits arginase activity (i.e., reducing urea production by 50%) without altering NOS activity, enhanced NO production (by 55%) from activated macrophages. The enhancement of NO production by l-norvaline was inversely related to the extracellular level ofl-arginine. A more pronounced increase in NO production was observed at the lower level of extracellular l-arginine, i.e., a 55 vs. 28% increase for 0.05 and 0.1 mM extracellularl-arginine, respectively. When the l-arginine concentration exceeded 0.5 mM, thel-norvaline effect was abolished. These results indicate that arginase can compete with NOS for their common substrate and thus inhibit NO production. This regulatory mechanism may be particularly important when the extracellular supply ofl-arginine is limited.