Lipid polymorphism was studied with the aim to gain more insight in bilayer to non-bilayer phase transitions, with particular emphasis on the development of cubic structures on one hand and inverted hexagonal structures on the other hand. Thin vitrified films prepared from aqueous lipid suspensions were used in this study. The entire hydrated contents of these films can be visualized in their two-dimensional projection by cryo-electron microscopy. As the starting material, unilamellar vesicles were prepared from mixtures of dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol. By heating of the suspension, vesicle fusion (Frederik et al. (1989) Biochim. Biophys. Acta 979, 275-278) and lipid polymorphism was induced. From these suspensions thin films were prepared at various temperatures, and vitrified for low temperature observation. In a parallel series of experiments samples were fast-frozen for freeze-fracture analysis. In vitrified thin films bilayer structures were often observed in coexistence with an inverted hexagonal structure. The bilayer areas were frequently of a complex structure because multiple contacts between stacked membranes were found. These contact points were variable in size and shape and usually had the form of a diabolo (when viewed side-on) giving the impression of a bilayer contact with an aqueous channel. This structure is compatible with the interlamellar attachment site (ILA) proposed by Siegel ((1986) Biophys. J. 49, 1155-1170). In some specimens ILA's seemed to merge into arrays. After thermal cycling of the suspension, arrays of packed globules were observed, which are likely the result of close packing of ILA's. The arrays probably represent a cubic structure. A comparison of freeze-fracture replicas and vitrified thin films indicated that both techniques may provide valuable structural information on lipid polymorphism. Most of the lipidic particles observed by freeze-fracturing probably correspond to the ILA's (fractured around their waist region) as observed in vitrified thin films. The results obtained with vitrified thin films were interpreted in relation to the principles of thin-film formation. Finally, we speculate that lipid structures occurring close to each other in space may represent a developmental series of structures occurring successively in time.