Glycoproteins of molecular weights (MW) of 38,000, 32,000, and 26,000 are found in surfactant isolated from rat lungs. These proteins were further examined for their specificity to pulmonary surfactant, structural and metabolic interrelationships, and relation to the ontogenesis of pulmonary surfactant. With ultracentrifugations in salt and sucrose density gradients, a preparation of pulmonary surfactant was isolated from rat lung lavage fluid, which was rich in surfactant lipids (phosphatidylcholine and phosphatidylglycerol), and contained exclusively the 38,000-, 32,000-, 26,000-, and 10,000- to 12,000-dalton proteins. The 38,000-, 32,000-, and 26,000-dalton proteins are not serum proteins. Using an antiserum specific for the combined 38,000-, 32,000-, and 26,000-dalton proteins and the immunoperoxidase technique, the source of one or more of these three proteins was found to be alveolar epithelial type II cells, the cells involved in the synthesis and secretion of pulmonary surfactant. These proteins, when dissociated from lipids, show considerable self-association and form homopolymers. On isoelectric focusing, these proteins show considerable charge heterogeneity, which, in large part, is due to terminally linked sialic acid residues. Partial proteolysis of these proteins and subsequent analyses of the released polypeptides suggest the existence of large segments of homology between the 38,000-, and 32,000-dalton proteins. The relationship of the 38,000-, and 32,000-dalton proteins with the 26,000-, and 10,000- to 12,000-dalton proteins is not clear as yet. The results of protein analyses of purified tubular myelin and of lamellar bodies suggest that the 26,000-dalton protein may be derived extracellularly, possibly from other surfactant proteins by the action of enzymes present in the alveolar lining layer. We observed no reactivity of the antibody raised against the 38,000-, 32,000-, and 26,000-dalton proteins with the 10,000- to 12,000-dalton protein. The 38,000-, 32,000-, and 26,000-dalton proteins appear during fetal lung maturation at the same gestational time as the surfactant is known to appear, and their combined content increases thereafter in fetal lungs and in amniotic fluid. It appears that the less glycosylated (32,000-dalton protein) of the 38,000- and 32,000-dalton proteins appears first during fetal lung development.