The primary goal of this study was to investigate the effects of glucose infusion on surfactant phosphatidylcholine (PC) metabolic kinetics in the lungs. A new stable isotope tracer model was used in which [1,2-¹³C₂]acetate and uniformly labeled [U-¹³C₁₆]palmitate were infused in 12 normal overnight-fasted pigs to quantify lung surfactant kinetics with or without glucose infusion (24 mg · kg¹ · min¹). With glucose infusion, the rate of surfactant PC incorporation from de novo synthesized palmitate increased from the control value of 2.1 ± 0.2 to 15.5 ± 1.9 nmol PC-bound palmitate · h¹ · g wet lung¹ (P < 0.05), whereas the incorporation rate from plasma preformed palmitate decreased from the control value of 20.9 ± 1.9 to 11.6 ± 1.1 nmol palmitate · h¹ · g wet lung¹ (P < 0.05). The palmitate composition in lamellar body surfactant PC increased from the control value of 61.7 ± 2.1% to 75.9 ± 0.6% (P < 0.05). The surfactant PC secretion rate decreased from the control value of 239.0 ± 26.1 to 81.9 ± 5.3 nmol PC-bound palmitate · h¹ · g wet lung¹ (P < 0.05). We conclude that, whereas surfactant secretion was inhibited by glucose infusion, neither total surfactant PC synthesis nor the surfactant PC pool size was significantly affected due to an increased reliance on de novo synthesized fatty acids.