Both surfactant protein (SP) D and granulocyte-macrophage colony-stimulating factor (GM-CSF) influence pulmonary surfactant homeostasis, with the deficiency of either protein causing marked accumulation of surfactant phospholipids in lung tissues and in the alveoli. To assess whether the effects of each gene were mediated by distinct or shared mechanisms, surfactant homeostasis and lung morphology were assessed in 1) double-transgenic mice in which both SP-D and GM-CSF genes were ablated [SP-D(−/−),GM(−/−)] and 2) transgenic mice deficient in both SP-D and GM-CSF in which the expression of GM-CSF was increased in the lung. Saturated phosphatidylcholine (Sat PC) pool sizes were markedly increased in SP-D(−/−),GM(−/−) mice, with the effects of each gene deletion on surfactant Sat PC pool sizes being approximately additive. Expression of GM-CSF in lungs of SP-D(−/−),GM(−/−) mice corrected GM-CSF-dependent abnormalities in surfactant catabolism but did not correct lung pathology characteristic of SP-D deletion. In contrast to findings in GM(−/−) mice, degradation of [³H]dipalmitoylphosphatidylcholine by alveolar macrophages from the SP-D(−/−) mice was normal. The emphysema and foamy macrophage infiltrates characteristic of SP-D(−/−) mice were similar in the presence or absence of GM-CSF. Taken together, these findings demonstrate the distinct roles of SP-D and GM-CSF in the regulation of surfactant homeostasis and lung structure.