We have previously demonstrated that very premature infants receiving glucose at 17 μmol/kg min plus appropriate supply of parenteral lipids (Intralipid®) and amino acids (TrophAmine®) maintained normoglycemia by glucose produced primarily via gluconeogenesis. The present study addressed the individual roles of parenteral lipids and amino acids in supporting gluconeogenesis. Fourteen premature infants (993 ± 36 g 27 ± 1 wk) (mean ± SE) were studied for 8 h on d 5 ± 1 of life. All infants were receiving standard TPN prior to the study. At start of study, the glucose infusion rate was decreased to ∼17 μmol/kg min and either Intralipid® (g + AA; n = 8) or TrophAmine® (g + IL; n = 6) was discontinued. Data from 14 previously studied infants receiving glucose (∼17 μmol/kg min) + TrophAmine® + Intralipid® (g + AA + IL) are included for comparison. Gluconeogenesis was measured by [U-13 C]glucose, (g + AA) and (8 infants of the g + AA + IL group) or [2-13C]glycerol, (g + IL) and (6 infants of the g + AA + IL group). Infants studied by the same method were compared. Withdrawal of Intralipid® resulted in decreased gluconeogenesis, 6.3 ± 0.9 (g +AA) vs. 8.4 ± 0.7 μmol/kg min (g + AA + IL) (p = 0.03). Withdrawal of TrophAmine® affected neither total gluconeogenesis, 7.5 ± 0.8 vs. 7.9 ± 0.9 μmol/kg min nor gluconeogenesis from glycerol, 4.4 ± 0.6 vs. 4.9 ± 0.7 μmol/kg min (g+ IL and g + AA + IL groups, respectively). In conclusion, in parenterally fed very premature infants, lipids play a primary role in supporting gluconeogenesis.