SP-B is the protein in pulmonary surfactant with the greatest capacity to augment the phospholipids, ability to resist surface tension, and capability to prevent collapse of pulmonary alveoli. Synthetic peptides derived from the structure of SP-B and simplified analogues of these SP-B-derived peptides were found by tryptophan fluorescence to partition within the phospholipid layer in contact with both polar head groups and acyl side chains of the phospholipids. The intermittent hydrophilic basic residues were found to be essential for full activity, probably because of electrostatic interactions formed with phosphates of the polar head groups. The hydrophobic stretches of residues in SP-B and the related peptides supplement the activity through interaction with the phospholipid acyl side chains. By increasing intermolecular and intramolecular order of the phospholipid layer and thereby stability of the layer, the SP-B analogues provide strong surfactant activity. Simplified peptide analogues of SP-B, dispersed in DPPC and POPG, provide strong surfactant activity in vitro and in the lungs of premature infant rabbits, rhesus monkeys, and humans.