It is known that cells and organisms can indirectly ``sense{''} changes in l-arginine availability via changes in the activity of various metabolic pathways. However, the mechanism(s) by which genes can be directly regulated by l-arginine in mammalian cells have not yet been elucidated. We investigated the effect of l-arginine in the in vivo model of peritoneal inflammation in mice and in vitro in RAW 264.7 macrophages. A detailed analysis of basic physiological functions and selected intracellular signaling cascades revealed that l-arginine is crucial for the acceleration of macrophage activation by bacterial lipopolysaccharide. l-arginine increased the production of reactive oxygen species, nitric oxide, release of Ca2+, as well as inducible nitric oxide synthase expression. Interestingly, the effect of l-arginine on macrophage activation was dependent on the phosphorylation of mitogen-activated protein kinases and activity of phospholipase C. In RAW 264.7 cells, l-arginine was shown to modulate the response of macrophages toward lipopolysaccharide via the activation of G-protein-coupled receptors. According to our data, we concluded that l-arginine availability plays a key role in the initiation of intracellular signaling pathways that trigger the lipopolysaccharide-induced inflammatory responses in murine macrophages. Although macrophages are partially stimulated in the absence of extracellular l-arginine, the presence of this amino acid significantly accelerates the sensitivity of macrophages to bacterial endotoxin.