Background: Animal-derived surfactants containing surfactant proteins B (SP-B) and C (SP-C) are used to treat respiratory distress syndrome (RDS) in preterm infants. SP-B (79 residues) plays a pivotal role in lung function and the design of synthetic lung surfactant. Super Mini-B (SMB), a 41-residue peptide based on the N- and C-domains of SP-B joined with a turn and two disulfides, folds as an α-helix hairpin mimicking the properties of these domains in SP-B. Here, we studied ‘B-YL’, a 41-residue oxidation-resistant SMB variant that has its four Cys and two Met residues replaced by Tyr and Leu, respectively, to test whether these hydrophobic substitutions produce a surface-active, α-helix hairpin.Methods:Structure and function of B-YL and SMB in surfactant lipids were compared with CD and FTIR spectroscopy and molecular dynamic (MD) simulations, and surface activity with captive bubble surfactometry and in lavaged, surfactant-deficient adult rabbits.Results:CD and FTIR spectroscopy of B-YL in surfactant lipids showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to SMB in lipids. MD simulations confirmed that B-YL maintained its α-helix hairpin in a lipid bilayer, matching the hairpin obtained from MD of SMB. Unlike the disulfide-reinforced helix-turn of SMB, the B-YL fold was stabilized by a core of clustered Tyr linking the N- and C-helices through noncovalent interactions involving aromatic rings. B-YL in surfactant lipids demonstrated excellentin vitrosurface activity and good oxygenation and dynamic compliance in lavaged, surfactant-deficient adult rabbits.Conclusions:‘Sulfur-free’ and ‘oxidation-resistant’ B-YL forms an amphipathic helix-hairpin in surfactant liposomes with high surface activity and is functionally similar to SMB and native SP-B. B-YL’s resistance against free oxygen radical damage provides an extra edge over oxidized SMB in the treatment of respiratory failure in preterm infants with RDS and children and adults with acute lung injury.