Surfactant reduces surface tension at pulmonary air-liquid interfaces. Although its major component is dipalmitoyl-phosphatidylcholine (PC16:0/16:0), other PC species, principally palmitoylmyristoyl-PC, palmitoylpalmitoleoyl-PC, and palmitoyloleoyl-PC, are integral components of surfactant. The composition and metabolism of PC species depend on pulmonary development, respiratory rate, and pathologic alterations, which have largely been investigated in animals using radiolabeled precursors. Recent advances in mass spectrometry and availability of precursors carrying stable isotopes make metabolic experiments in human subjects ethically feasible. We introduce a technique to quantify surfactant PC synthesis in vivo using deuteriated choline coupled with electrospray ionization tandem mass spectrometry. Endogenous PC from induced sputa of healthy volunteers comprised 54.0 ± 1.5% PC16:0/16:0, 9.7 ± 0.7% palmitoylmyristoyl-PC, 10.0 ± 1.0% palmitoylpalmitoleoyl-PC, and 13.1 ± 0.3% palmitoyloleoyl-PC. Infusion of deuteriated choline chloride (3.6 mg/kg body weight) over 3 hours resulted in linear incorporation into PC over 30 hours. After a plateau of 0.61 ± 0.04% labeled PC between 30 and 48 hours, incorporation decreased to 0.30 ± 0.02% within 7 days. Compared with native PC, fractional label was initially lower for PC16:0/16:0 (31.9 ± 8.3%) but was higher for palmitoyloleoyl-PC (21.0 ± 1.2%), and equilibrium was achieved after only 48 hours. We conclude that infusion of deuteriated choline and electrospray ionization tandem mass spectrometry is useful to investigate surfactant metabolism in humans in vivo.