The kidney is a major site of arginine synthesis where citrulline is converted to arginine via argininosuccinate synthase (ASS) and lyase (ASL). The rate-limiting step in arginine synthesis by the normal kidney is the rate of citrulline delivery and uptake to the renal cortex. We tested whether with chronic kidney disease (CKD) renal arginine synthesis may be compromised. Using the 5/6 renal ablation/infarction (A/I) injury model, we measured renal citrulline delivery and uptake as well as arginine release at early, moderate, and severe stages of CKD vs. healthy controls. The renal plasma flow (RPF) and arterial-renal venous difference was measured at baseline and during citrulline infusion. Citrulline delivery was reduced at all stages of disease due to marked reductions in RPF and despite moderately increased plasma citrulline. Early after 5/6 A/I, the kidney demonstrated a compensatory increase in citrulline uptake while at moderate and severe injury baseline citrulline uptake fell. At all stages of CKD, renal arginine release was markedly reduced. Citrulline infusion increased plasma citrulline in all groups, resulting in increased renal delivery vs. baseline. In healthy kidneys and early injury, citrulline uptake increased with the infusion, but only in the normal kidney did arginine production increase in parallel with the increased citrulline uptake. At moderate and severe injury, there was no increase in citrulline uptake or arginine production. The fall in arginine production in 5/6 A/I was due to an early loss of ASS and ASL conversion of citrulline, which combined with a later reduction in citrulline uptake.