In preterm infants, both hypo- and hyperglycemia are a frequent problem. Intravenous lipids can affect glucose metabolism by stimulation of gluconeogenesis by providing glycerol, which is a gluconeogenic precursor, and/or free fatty acids (FFA), which are stimulants of the rate of gluconeogenesis. In 25 preterm infants, glucose production and gluconeogenesis were measured using stable isotope techniques during a 6-h infusion of glucose only, glucose plus glycerol, or glucose plus an intravenous lipid emulsion. Two lipid emulsions differing in FFA composition were used: Intralipid (∼60% polyunsaturated FFA) and Clinoleic (∼60% monounsaturated FFA). The rate of glucose infusion was 22 μmol·kg⁻¹·min⁻¹in all groups. During the study infusion, the FFA concentrations were higher in both lipid groups vs. the glycerol group ( P < 0.001). Compared with baseline, the glucose production rate increased in the Intralipid group, whereas it decreased in the other groups ( P = 0.002) due to a significant increase in gluconeogenesis in the Intralipid group ( P = 0.016). The plasma glucose concentration was significantly higher during Intralipid infusion vs. the other groups ( P = 0.046). Our conclusion was that Intralipid enhanced glucose production by increasing gluconeogenesis in preterm infants. This can be ascribed to the stimulatory effect of FFA in addition to any effect of glycerol alone. The lack of stimulation of gluconeogenesis in the Clinoleic vs. the Intralipid group suggests that different classes of fatty acids exert different effects on glucose kinetics in preterm infants.