Surfactant proteins B and C (SP-B and SP-C), together with phospholipids, are important constituents of pulmonary surfactant and of preparations used for treatment of respiratory distress syndrome (RDS). SP-B belongs to the saposin family of homologous proteins, which include other lipid-interacting proteins, like the membranolytic NK-lysin. SP-B, in contrast to other saposins, is hydrophobic and a disulfide-linked dimer, and its mechanism of action is not known. A model of the three-dimensional structure of one SP-B subunit was generated from the structure of monomeric NK-lysin determined by nuclear magnetic resonance, and the SP-B dimer was formed by joining two subunits via the intersubunit disulfide bond Cys48-Cys48'. After energy minimization, intersubunit hydrogen bonds/ion pairs were formed between the strictly conserved residues Glu51 and Arg52, which creates a central non-polar region located in between two clusters of positively charged residues. The structural features support a function of SP-B in cross-linking of lipid membranes. Mixtures of phospholipids, an SP-C analogue and polymyxin B (which cross-links lipid vesicles but is structurally unrelated to SP-B) exhibit in vitro surface activity which is indistinguishable from that of analogous mixtures containing SP-B instead of polymyxin B. This suggests an avenue for identification of SP-B analogues that can be used in synthetic surfactants for treatment of RDS.