Examination of lung washings from primitive air-breathing fishes (ropefish, bichirs, and gar) revealed a lipid-based surfactant with an average disaturated phospholipid-to-total phospholipid ratio five times lower than in mammals. The lung lavage of fishes was exceptionally rich in cholesterol, resulting in average cholesterol-to-phospholipid ratios three times higher, and cholesterol-to-disaturated phospholipid ratios nearly 15 times higher, than those of mammals. Removal of lung surfactant doubled the pressures necessary to initially open the anterior regions of collapsed lungs in all three fish species but had little or no effect on pressures required to fill the lung (i.e., compliance) after the initial opening. The elevated cholesterol content found in pulmonary surfactant of these fishes is consistent with such findings in other ectotherms, suggesting that the proportional elevation of cholesterol may serve to stabilize the fluidity of the lung surfactant over broader temperature ranges. The influence of surfactant on lung opening pressures rather than on compliance contrasts with that seen in mammals and supports an "antiglue" role of pulmonary surfactant in the simpler open-design lungs of lower vertebrates.