Renal arginine synthesis is a major endogenous source of arginine. Argininosuccinate lyase occurs almost exclusively in kidney cortex. In studies with isolated renal cortical tubules, we observed rapid rates of arginine synthesis from citrulline, provided a source of the N atom of the guanidino group of arginine was supplied. Aspartate, glutamate, or glutamine were effective, whereas glycine, alanine, serine, or NH4Cl were ineffective as this second substrate. Arginine synthesis as a function of citrulline concentration was determined and was found to be highly sensitive to citrulline concentrations in the physiological range (60 microM), suggesting that renal arginine synthesis in vivo could be regulated by circulating citrulline levels. Therefore, arginine synthesis by the kidney was investigated in vivo by measuring the net flux of citrulline and arginine in saline-infused (control group) and citrulline-infused rats. In normal animals, uptake of citrulline was 60.5 +/- 20.7 nmol.min-1.100 g body wt-1, and a similar arginine release was observed. Citrulline infusion that increased circulating citrulline levels fourfold resulted in a similar increase in renal citrulline uptake (224 +/- 33 nmol.min-1.100 g-1) and a similar increase in renal production of arginine. The results suggest that the availability of citrulline is a limiting factor for renal arginine synthesis in rats.