Surfactant protein B (SP-B), a peptide found in organic solvent extracts of mammalian surfactant, has been isolated from surfactant previously by column chromatography and/or preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE). We have developed a method for isolation of SP-B from bovine surfactant utilizing differential organic extraction. Dried surfactant, isolated from lavage of excised cow lungs, was delipidated by extraction with diisopropyl ether-butanol (3:2). The aqueous layer, containing surfactant proteins, was dried and then was sequentially extracted with diethyl ether-ethanol (3:1) and CHCl3:MeOH:HCl (3:2:0.005 N). SP-B partitioned into chloroform-methanol, which was evaporated under N2. Purified SP-B, quantitated by Coomassie dye binding, represented 1% (wt/wt) of the original surfactant with a final phospholipid-to-protein ratio less than 1. Silver-stained SDS/PAGE of the SP-B extract revealed a single band at 9 kDa (reduced) and 18 kDa (nonreduced), which by immunoblotting reacted strongly with monospecific anti-SP-B antibody. Amino acid sequence analysis confirmed the presence of NH2 and N-1 terminal sequences of bovine SP-B. This procedure offers a rapid, reliable method for isolation of purified SP-B from whole surfactant.