Pulmonary surfactant is synthesized and secreted by alveolar type II cells. Radioactive phosphatidylcholine has been used as a marker for surfactant secretion. We report findings that suggest that surfactant inhibits secretion of 3H-labeled phosphatidylcholine by cultured rat type II cells. The lipid components and the surfactant protein group of Mr 26,000-36,000 (SP 26-36) inhibit secretion to different extents. Surfactant lipids do not completely inhibit release; in concentrations of 100 micrograms/ml, lipids inhibit stimulated secretion by 40%. SP 26-36 inhibits release with an EC50 of 0.1 microgram/ml. At concentrations of 1.0 microgram/ml, SP 26-36 inhibits basal secretion and reduces to basal levels secretion stimulated by terbutaline, phorbol 12-myristate 13-acetate, and the ionophore A23187. The inhibitory effect of SP 26-36 can be blocked by washing type II cells after adding SP 26-36, by heating the proteins to 100 degrees C for 10 min, by adding antiserum specific to SP 26-36, or by incubating cells in the presence of 0.2 mM EGTA. SP 26-36 isolated from canine and human sources also inhibits phosphatidylcholine release from rat type II cells. Neither type I collagen nor serum apolipoprotein A-1 inhibits secretion. These findings are compatible with the hypothesis that surfactant secretion is under feedback regulatory control.