Hepatocytes can be stimulated to express high levels of inducible nitric oxide synthase (iNOS), which utilizes arginine for nitric oxide (NO) synthesis. Hepatocytes also synthesize and catabolize arginine, an intermediate in the urea cycle, raising the possibility that the urea pathway may provide substrate for hepatic NO synthesis. To identify the sources of arginine for iNOS, we measured the release of NO-2 + NO-3 and urea in isolated rat livers perfused in a recirculation model with a Krebs-Henseleit-bicarbonate buffer containing either no added amino acid, arginine, or precursors for urea synthesis. To induce iNOS expression, rats were injected with killed Corynebacterium parvum (C. parvum) or with endotoxin. In livers from C. parvum- and endotoxin-treated rats, we found that 1) an intracellular source of arginine exists that provides substrate to iNOS; 2) additional exogenous arginine increase NO synthesis, demonstrating that endogenous arginine is insufficient for maximal NO synthesis; and 3) an increase in the rate of endogenous arginine synthesis within the urea cycle is inefficient in increasing NO synthesis, demonstrating the independence of the two pathways in the liver.