Background: Citrulline, a nonprotein amino acid, is an intermediate of the urea cycle and synthesized in small intestine. Lower plasma citrulline levels were associated with reduced function of enterocytes. Necrotizing enterocolitis (NEC) causes high morbidity and mortality, and leads impaired intestinal functions. Methods: Plasma citrulline levels of neonates with a gestational age <32 weeks and 1,500 gm who developed NEC stage II/III were measured by high-performance liquid chromatography. Results: We enrolled 36 preterm infants including 20 with NEC and 16 controls. Median citrulline levels of NEC and control groups were 8.6 and 20.18 mol/l (P < 0.05), and cut off level of citrulline was 13.15 mol/l with a sensitivity of 80% and a specificity of 82%. Median arginine levels of NEC and control groups were 22.02 and 39.89 mol/l (P < 0.05), and cut off level of arginine was 28.52 mol/l with a sensitivity of 70% and a specificity of 75%. Blood sampling day, gender, parenteral, and enteral nutrition did not affect the amino acid levels. Conclusion: We found lower plasma citrulline and arginine levels in preterm infants with NEC. Further studies are needed to determine most appropriate levels to predict recovery and prognosis of NEC, and treatment options with these amino acids in preterm infants. (C) 2013 Wiley Periodicals, Inc.