Rat peritoneal macrophages were incubated in the presence of 0.05-1.0 mM-[14C]citrulline. The synthesis of [14C]arginine from 0.1 mM-[14C]citrulline was about 300 pmol/h per 10(6) cells in macrophages from saline-injected (control) rats. Both arginine synthesis from citrulline and nitrate production (an indicator of NO generation) were increased about 3-fold in the cells from lipopolysaccharide (LPS)-treated animals. The arginine synthesis was very sensitive to extracellular citrulline concentration in the range found in plasma (0.05-0.1 mM). The rate of arginine synthesis from citrulline was inhibited by about 20% by 0.5 mM-L-glutamine in both control and LPS-treated rat cells, but was inhibited by 0.5 mM-L-arginine only in control cells. Our results demonstrate that citrulline, produced by NO synthetase, can be recycled to arginine in macrophages. The citrulline-arginine cycle may contribute to the regulation of intracellular availability of arginine and thus the prolonged production of NO by macrophages.