Lung structure and function vary widely among vertebrates. Despite their diversity, all lungs are internal, fluid-lined structures that change volume and hence face similar biophysical problems. For example, if the surface tension of the fluid lining is high, this may lead to collapse or flooding of the lung In mammals, these problems are largely overcome by the presence of a mixture of surface-active lipids and proteins (pulmonary surfactant), which lowers the surface tension of the fluid lining, particularly at very low lung volumes. This action is due primarily to a disaturated phospholipid (DSP), predominantly dipalmitoylphosphatidylcholine (DPPC), which exists in the ordered, gel state below 41°C Cholesterol (CHOL) and unsaturated phospholipids (USPs) promote respreading upon inflation by converting DPPC to the disordered, liquid-crystalline state. It appeared to us that a DSP-rich surfactant, with its high phase transition temperature, is likely to be of only limited use in the lungs of ectothermic vertebrates that have body temperatures between 20° and 30°C We determined the presence and composition of surfactant in species from a range of vertebrate taxa maintained at 23°C and related variations in phospholipid (PL) head groups, CHOL/PL, DSP/PL, and CHOL/DSP to lung structure and function, phylogeny, and environmental selection pressures such as body temperature. All air breathers examined had a pulmonary surfactant containing USP, DSP, and CHOL. In general, mammals had greater amounts of surfactant lipids than did most nonmammals when expressed per gram of wet lung mass (g WL). However, when expressed per unit of respiratory surface area (cm² RSA), most nonmammalian species tested had six- to 30-fold greater amounts of surfactant lipid than did mammals. Phosphatidylcholine was the predominant PL, and only the minor phospholipids varied between species. We observed surfactant to change in composition from a mixture of very high CHOL/very low DSP in primitive air-breathing actinopterygiian fish, to intermediate CHOL/intermediate DSP in derived lung, flsh and amphibians, to low CHOL/high DSP in reptiles and mammals. We have also observed smaller changes in surfactant composition between species and within individuals, which correlated with dfferences in body temperature, lifestyle, and lung maturity as well as with structure and function of the lung. We determined the pressure required to open a collapsed lung both before and after the removal of surfactant in several species of each vertebrate group and found in virtually all cases that surfactant functioned to lower the lung opening pressure. These findings were consistent with the surfactant functioning as an antiglue in these vertebrate groups. Possibly, acting as an antiglue represents the primitive function of surfactant. On the basis of the two apparently distinct types of surfactant composition, a high CHOL/ low DSP mixture in the primitive air-breathing fish and a mixture of low to intermediate CHOL and intermediate to high DSP levels in the derived sarcopterygiians and the tetrapods, we suggest that the CHOL -enriched surfactant may represent the primitive surfactant, or protosurfactant.