Deterioration of pulmonary surfactant function has been reported in interstitial lung disease; however, the molecular basis is presently unclear. We analyzed fatty acid (FA) profiles of several surfactant phospholipid classes isolated from large-surfactant aggregates of patients with idiopathic pulmonary fibrosis (IPF; n = 12), hypersensitivity pneumonitis (n = 5), and sarcoidosis (n = 12). Eight healthy individuals served as controls. The relative content of palmitic acid in phosphatidylcholine was significantly reduced in IPF (66.8 ± 2.5%; means ± SE; P < 0.01) but not in hypersensitivity pneumonitis (78.5 ± 1.8%) and sarcoidosis (78.2 ± 3.1%; control 80.1 ± 0.7%). In addition, the phosphatidylglycerol FA profile was significantly altered in the IPF patients, with a lower relative content of its major FA, oleic acid, at the expense of saturated FA. In the phosphatidylcholine class, a significant correlation between the impairment of biophysical surfactant function and decreased percentages of palmitic acid was noted. We conclude that significant alterations in the FA profile of pulmonary surfactant phospholipids occur predominantly in IPF and may contribute to the disturbances of alveolar surface activity in this disease.