In bullfrog (Rana catesbeiana) tadpoles, the lung begins to function at an advanced stage of metamorphosis. As a preliminary step for investigation of the mechanisms involved in lung maturation, pulmonary surfactant was prepared from tadpoles at advanced stages of metamorphosis and its biochemical properties were analyzed. Surfactant phospholipid analysis revealed that the major constituent was phosphatidylcholine (PC), as examined in the animals at late climax (stage 24). Other detectable phospholipids were phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylglycerol, a marker lipid in mammalian surfactant. As in mammals, PC in the surfactant was rich in saturated fatty acids, about 50% of fatty acid moieties being palmitic acid. The content of surfactant PC in the lung increased moderately around mid-climax and markedly at the end of climax. The effect of antiserum against bullfrog prolactin (PRL) on the pulmonary surfactant was studied in climactic tadpoles. The content of surfactant PC in the lung of the antiserum-treated larvae was lower than that in the lung of the normal rabbit serum-injected larvae, whereas the content of PC in the whole lung did not differ between the antiserum-treated and control groups. The results suggest that synthesis of surfactant in the amphibian lung is enhanced as metamorphosis progresses and that PRL is involved in lung maturation.