Inducible NO synthase (iNOS) and its generation of NO from l-arginine are subject to transcriptional as well as posttranscriptional control by cytokines. In this study, we describe a novel, translational mechanism of iNOS regulation by arginine availability. Using mouse inflammatory peritoneal macrophages stimulated with IFN-γ plus LPS, we demonstrate that the suppression of iNOS protein, which is observed after a 16-h (but not after a 6-h) pretreatment with IL-13, despite an unaltered iNOS mRNA level, results from arginine depletion by arginase. The addition of arginase inhibitors (in the pretreatment phase) or of arginine (in the stimulation phase) completely blocked the down-regulation of iNOS protein by IL-13. The rescuing effect of arginine supplementation was not due to a positive feedback regulation of iNOS expression via enhanced production of NO. A striking suppression of iNOS protein (but not of iNOS mRNA) was also seen, when IL-13 was replaced by purified arginase or when macrophages were stimulated with IFN-γ/LPS in arginine-free medium. Arginine deficiency specifically impaired the de novo synthesis and the stability of iNOS protein, but did not affect the production of TNF and the overall protein synthesis of the macrophages. From these results, we conclude that arginine not only functions as a substrate for iNOS, but is also critical for maintaining normal levels of iNOS protein in cytokine-stimulated macrophages.