A procedure is described for the isolation of a surface-active fraction from dog lung. This material meets the established criteria for pulmonary surfactant. The fraction was shown to contain lipid, protein, and carbohydrate. The predominant lipid present was dipalmitoyl phosphatidylcholine. Surface chemistry studies indicated the surface properties of the fraction could not be explained solely from a consideration of the properties of dipalmitoyl phosphatidylcholine. Electron microscopic studies demonstrated the presence of intact osmiophilic bodies as well as other myelin forms in the surface-active fraction. It is speculated that, in situ, the alveolar lining layer is similar to a structured gel.