Pulmonary surfactant conventionally is prepared from material obtained by endobronchial lavage. Although it has been assumed that the components of surfactant are secreted by alveolar type II cells, direct proof of this assumption has not been available. Furthermore, it is possible that the final material obtained by lavage has been modified after secretion or altered during the isolation procedure. It has been shown previously that type II cells, after 1 day in primary culture, secrete saturated phosphatidylcholine, one of the lipid components of surfactant. Because saturated phosphatidylcholine is not unique to surfactant and because type II cells in culture lose differentiated characteristics over the first several days in culture, it has not previously been established how closely the secretory products of cultures of type II cells resemble surfactant as obtained by endobronchial lavage. We therefore studied the morphologic, physical and chemical characteristics of the material that type II cells secrete under basal conditions and after stimulation with terbutaline or 12-O-tetradecanoyl-13-phorbol acetate. The secreted material resembled surfactant obtained by lavage; it was similar morphologically to the lamellar material and tubular myelin seen in the fluid-filled alveoli of fetal rats, it lowered surface tension to 5 mN per meter, and it contained the 72000 dalton apolipoprotein of surfactant (as measured by the 'rocket' immunoelectrophoresis technique). When cells were incubated for 22 h with [1-(14)C]acetate, the distribution of radioactivity in the secreted material was very similar to the phospholipid composition of rat surfactant. We conclude that the material secreted by alveolar type II cells after 1 day in primary culture is similar to surfactant obtained by endobronchial lavage.