As gut immaturity precludes full enteral feeding, very low birth weight (VLBW) preterm infants receive parenteral nutrition (PN) during the first few weeks of life. Weaning VLBW infants off PN, however, is a top priority since PN is associated with a high risk of complications. The decision making is purely empirical, as there is currently no suitable index of gastrointestinal (GI) maturity. Plasma citrulline concentration is considered an index of GI function in conditions such as short-bowel syndrome and coeliac disease in adults. To identify the factors determining urinary citrulline excretion, and determine whether urinary citrulline excretion could be used as a non-invasive index of GI tolerance to enteral feeding, nutritional intake and urinary citrulline were monitored bi-weekly in forty-seven preterm infants < 1500g (interquartiles 880-1320g), during their stay in the Neonatology unit. Median urinary citrulline was 24-7 mu mol/mmol creatinine (14-5-38-6 mu mol/mmol creatinine). No relationship was observed with the percentage of energy tolerated enterally. In multivariate regression analysis, weak correlations were found with post-conceptional age (P=0-001), parenteral amino acid supply (P=0.001) and the daily volume of enteral mixture administered (P=0.043). A significant correlation was found with urinary nitrite + nitrate excretion (r 0.47; P<0-001). We conclude that in preterm infants: (1) one of the major determinants of urinary citrulline may be the biosynthesis of citrulline from arginine by NO-synthase; (2) urinary citrulline cannot be used to predict GI tolerance. This is consistent with the observations that, in neonatal gut, citrulline is converted to arginine in situ rather than exported towards the kidneys as observed in adults.