Abstract The hypothesis was tested that induction of arginase expression in macrophages (Mφ) diminishes nitric oxide (NO) synthesis due to intracellular competition between arginase and inducible nitric oxide synthase (iNOS) for L-arginine (L-arg). Murine Mφ cell lines and bone marrow-derived Mφ (BMM) were stimulated to express either 1NOS or arginase or to coexpress these two enzymes. The response pattern obtained was complex but allowed the following conclusions: (1) 1NOS and arginase are differentially regulated. (ii) High intracellular arginase levels do not limit the capacity of Mφ to synthesize NO even when the L-arg concentration in the culture medium is lowered to physiological levels. (iii) Arginase levels in BMM pre-exposed to either Mφ colony-stimulating factor (M-CSF) or granulocyte-Mφ colony-stimulating factor (GM-CSF) differ markedly, but 1NOS expression and NO synthesis by the two BMM types is similar. (iv) Regulation of 1NOS and arginase differs between primary murine bone marrow Mφ and murine Mφ cell lines. (v) Arginase activity appears to be inhibited during high-output NO synthesis. Taken together, our results show that NO production by Mφ is not compromised by conditions that increase intracellular arginase activity.