Mice made granulocyte macrophage-colony stimulating factor (GM-CSF)-deficient by homologous recombination maintain normal steady-state hematopoiesis but have an alveolar accumulation of surfactant lipids and protein that is similar to pulmonary alveolar proteinosis in humans. We asked how GM-CSF deficiency alters surfactant metabolism and function in mice. Alveolar and lung tissue saturated phosphatidylcholine (Sat PC) were increased six- to eightfold in 7- to 9-wk-old GM-CSF-deficient mice relative to controls. Incorporation of radiolabeled palmitate and choline into Sat PC was higher in GM-CSF deficient mice than control mice, and no loss of labeled Sat PC occurred from the lungs of GM-CSF-deficient mice. Secretion of radiolabeled Sat PC to the alveolus was similar in GM-CSF-deficient and control mice. Labeled Sat PC and surfactant protein A (SP-A) given by tracheal instillation were cleared rapidly in control mice, but there was no measurable loss from the lungs of GM-CSF-deficient mice. The function of the surfactant from GM-CSF-deficient mice was normal when tested in preterm surfactant-deficient rabbits. GM-CSF deficiency results in a catabolic defect for Sat PC and SP-A.