Pulmonary surfactant, a complex mixture of lipids and proteins, lowers the surface tension in terminal air spaces and is crucial for lung function. Within an animal species, surfactant composition can be influenced by development, disease, respiratory rate, and/or body temperature. Here, we analyzed the composition of surfactant in three heterothermic mammals (dunnart, bat, squirrel), displaying different torpor patterns, to determine: 1) whether increases in surfactant cholesterol (Chol) and phospholipid (PL) saturation occur during long-term torpor in squirrels, as in bats and dunnarts; 2) whether surfactant proteins change during torpor; and 3) whether PL molecular species (molsp) composition is altered. In addition, we analyzed the molsp composition of a further nine mammals (including placental/marsupial and hetero-/homeothermic contrasts) to determine whether phylogeny or thermal behavior determines molsp composition in mammals. We discovered that like bats and dunnarts, surfactant Chol increases during torpor in squirrels. However, changes in PL saturation during torpor may not be universal. Torpor was accompanied by a decrease in surfactant protein A in dunnarts and squirrels, but not in bats, whereas surfactant protein B did not change in any species. Phosphatidylcholine (PC)16:0/16:0 is highly variable between mammals and is not the major PL in the wombat, dunnart, shrew, or Tasmanian devil. An inverse relationship exists between PC16:0/16:0 and two of the major fluidizing components, PC16:0/16:1 and PC16:0/14:0. The PL molsp profile of an animal species is not determined by phylogeny or thermal behavior. We conclude that there is no single PL molsp composition that functions optimally in all mammals; rather, surfactant from each animal is unique and tailored to the biology of that animal. lung; temperature; surfactant proteins; electrospray ionization mass spectrometry; cholesterol PULMONARY SURFACTANT is a complex mixture of lipids (90% by weight) and proteins (10% by weight) that reduces surface tension in the terminal air spaces, thus preventing pulmonary collapse (39). Phospholipids (PL) comprise 80 -90% of the surfactant lipids, with phosphatidylcholine (PC) being the most abundant PL (70 -85%). In homeothermic mammalian surfactant, the disaturated molecular species (molsp), dipalmitoylphosphatidylcholine (PC16:0/16:0) is traditionally considered to be the major contributor to surfactant surface activity (39). The anionic PLs, phosphatidylglycerol (PG) and phosphatidylinositol (PI) contribute ϳ10% to the total PL (39). The neutral lipid, cholesterol (Chol), comprises 10 -20 mol% of the total lipid (39) and is thought to contribute to surfactant fluidity and thus, spreadability over the alveolar surface (39). There are four surfactant proteins (SP). SP-A and SP-D are hydrophilic, calcium-dependent, carbohydrate-binding proteins involved in lung defense and surfactant homeostasis (22). SP-B and SP-C are hydrophobic proteins (40) that facilitate the adsorption of lipids to the air-liquid interface a...