Pulmonary surfactant phospholipids may assume several different structures including tubular myelin, unilamellar and multilamellar vesicles, and others. These populations of materials appear to have similar phospholipid compositions but may differ in their association with surfactant proteins SP-A, SP-B, or SP-C. We have used electron microscopy to determine the changes in structure of simple lipid mixtures (phosphatidylglycerol, dipalmitoylphosphatidylcholine) produced by adding one or combinations of the three proteins. Adding SP-A to lipids generated multilamellar structures composed of membranes with fuzzy or particulate surfaces. In contrast, SP-B or SP-C generated discoidal particles and structures that appeared to be sheets of membrane formed by associated particles. Used together, SP-A and SP-B reorganized some of the lipid into tubular myelin, a structure that was not observed in SP-A, SP-C recombinants. These observations confirm the in vitro formation of tubular myelin reported by others and support the possibility that surfactant materials with defined structure can be produced in vitro for analyses of their molecular organizations.