Hydrophobic, small molecular weight, surfactant- associated protein of Mr=6000 (SAP-6) was isolated from bovine, canine, and human alveolar lavage and identified by silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. Lesser amounts of protein of Mr=14,000, 20,000, and 26,000 daltons also copurified with SAP-6, likely representing oligomers of the Mr=6,000 dalton protein. In the absence of sulfhydryl-reducing agents, increased amounts of the larger forms of the protein were observed. Antisera generated against bovine SAP-6 were used to further characterize the protein and distinguish it from the more abundant surfactant-associated glycoprotein of Mr=35,000 (SAP- 35) present in mammalian surfactants. Rabbit antisera generated against the bovine hydrophobic protein recognized SAP-6 and lesser amounts of the proteins of Mr=14,000, 20,000, and 26,000 daltons. The SAP-6 antisera were reactive against the hydrophobic proteins from human, bovine, and canine surfactants as assessed by immunoblot analysis after sodium dodecyl sulfate polyacrylamide gel electrophoresis. SAP-6 antisera did not detect bovine SAP-35 the abundant surfactant-associated glycoprotein, by immunoblot analysis; however, some reactivity of the anti-SAP-6 was detected against purified bovine SAP-35 by a sensitive enzyme-linked immune-adsorbant assay. Anti-SAP-6-did not react with bovine serum components either by immunoblot or by enzyme-linked immune- adsorbant assay. Monospecific antisera generated against bovine SAP-35 did not detect SAP-6 by immunoblot analysis. Immunoblot analysis of the protein in exogenous replacement surfactant preparations, Surfactant-TA and calf lung surfactant extract, utilizing anti-SAP-6 and anti-SAP-35 demonstrated that both preparations were enriched in SAP-6 and lacking in SAP-35. Surfactant-associated proteins, present in bovine lung-based replacement surfactants, are immunologically identified as SAP-6 and its oligomers.