L-arginine is the precursor of nitric oxide which plays an important role on pulmonary circulation and pulmonary vascular tone. Our aim was to compare the levels of L-arginine between infants with respiratory distress syndrome and infants without respiratory distress syndrome and to determine the relationship between plasma L-arginine concentrations and severity of disease. Thirty premature infants who were admitted to our neonatal intensive care unit were included the study Seventeen of these infants with respiratory distress syndrome were study group and the other 13 infants without respiratory distress syndrome served as controls. Blood collection was made before any treatment or intervention given to infants and tandem mass spectrometry was used for laboratory testing. In the respiratory distress syndrome group mean L-arginine level was 33.0 (+/- 11.5) mM/I, and in controls it was 79.0 (+/- 23) mM/I. This difference was statistically significant (P < 0.05). There was a reverse relationship between L-arginine levels and oxygenation index (r = 0.732, P = 0.001). If level of L-arginine is low or insufficient in respiratory distress syndrome patients' nitric oxide level would decrease in pulmonary circulation and results increased pulmonary resistance and severity of respiratory distress syndrome. We concluded that L-arginine levels are low in patients with respiratory distress syndrome and for further investigations, supplementation of respiratory distress syndrome patients with L-arginine may decrease disease severity.