Previous studies have demonstrated that lung-specific proteins are associated with surfactant lipids, particularly the highly surface active subfraction known as tubular myelin. We have isolated a surfactant-associated protein complex with molecular weight components of 36000, 32000, and 28000 and reassembled it withprotein-free lung surfactant lipids prepared as small unilamellar liposomes. The effects of divalent cations on the structure and surface activity of this protein-lipid mixture were investigated by following (1) thestate of lipid dispersion by changes in turbidity and by electron microscopy and (2) the ability of the surfactant lipids to form a surface film from an aqueous subphase at 37 C. The protein complex markedly increased the rate of Ca2+-induced surfactant-lipid aggregation. Electron microscopy demonstrated transformation of the small unilamellar liposomes (median diameter 440 A) into largeaggregates. The threshold Ca2+ concentration required for rapid lipid aggregation was reduced from 13 to 0.5 mM by the protein complex. This protein-facilitated lipid aggregation did not occur if Mg2+ was the only divalent cation present. Similarly, 5 mM Ca2+ but not 5 mM Mg2+ improved the ability of the protein-lipid mixture to form a surface film at 37 C. Extensive aggregation of the surfactant lipidswithout protein by 20 mM Ca2+ or 20 mM Mg2+ did not promote rapid surface film formation. These results add to thegrowing evidence that specific Ca2+-protein-lipid interactions are important in determining boththe structure and function of extracellular lung surfactant fractions.