The regulation of gluconeogenesis by low birth weight infants remains poorly delineated. To investigate this, 15 5-d-old infants (26-31-wk gestational age, 795-1485-g body weight), were infused i.v. for 4 h with uniformly labeled [U-13C]glucose (5 mg/kg/min) as the sole source of glucose. Intralipid (3 mg/kg/ min) was provided, but no amino acids were given. Blood samples were taken immediately before and after 4 h of tracer infusion. The isotopic enrichments of plasma glucose and alanine were measured by selected ion monitoring gas chromatography mass spectrometry. Glucose production rates were calculated from the isotopic dilution of plasma [U-13C]glucose and the glucose infusion rate. Gluconeogenesis was estimated from the relative isotopic enrichments of [M + 3]- and [M + 6]-glucose, using the isotopic enrichment of plasma [U-13C]alanine, to define the isotopic dilution of the 3-carbon pool, and previously published equations to calculate the isotopic dilution of hepatic oxaloacetate. Glucose production (15 +/- 9 mumol/kg/min) was negatively related to body weight (r = -0.67; p < 0.05) and the ratio of the isotopic enrichments of [13C3]alanine:[13C6]glucose (0.27 +/- 0.07) was positively related to body weight (r = 0.74; p < 0.025). Both relationships were exponential. Gluconeogenesis (via pyruvate) accounted for 72 +/- 28% of glucose production, and gluconeogenesis per unit body weight was negatively and exponentially (r = -0.82; p < 0.005) related to body weight. These results demonstrate that neonates whose birth weights are less than 1200 g have a particularly high glucose production rate secondary to enhanced gluconeogenesis. We speculate that these results reflect a high weight-specific glucose demand that is met by up-regulation of gluconeogenesis.