Surface-active material (SF) was isolated from human lung lavage fluid collected at autopsy employing differential and sucrose density gradient centrifugation. The isolated material showed well-defined electron microscopic structure, consisting of clearly preserved, closely packed vesicles with limiting membranes and inclusion bodies. It showed a very high degree of alkaline phosphatase specific activity and was devoid of other subcellular contaminants. The isolated material also showed a high phospholipid/protein ratio and increasing surface activity when monitored at different stages of purification. It contained 68.5% phosphatidylcholine, 11.5% phosphatidylglycerol and relatively smaller amounts of phosphatidylethanolamine and other individual phospholipid (PL) classes. In addition, cholesterol, unesterified fatty acids, triacylglycerols and other neutral lipids were found. Saturated fatty acids, particularly palmitic acid (16:0), predominated in the major PL fractions. However, various fatty acids of which oleic acid (18:1) constituted a large proportion also are present. Chemical analysis of the material showed that besides lipids and proteins, nucleic acids, sialic acid, hexose, amino sugars, nitrogen and phosphorus were present. The delipidated material showed the presence of three to four proteins as characterized by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, and gel permeation chromatography on Sephadex G-200 resolved two well-separated peaks. The first fraction contained serum-associated 68 kDa protein, while the second fraction had two apoproteins with molecular weights of 34 kDa and 10 kDa. These two proteins were associated with the SF and they, as well as the whole surface-active material, strongly reacted with the antibody directed against the whole SF in a double-diffusion immunoprecipitation assay.(ABSTRACT TRUNCATED AT 250 WORDS)