Multilamellar stacking seen in negatively stained lipid vesicle-tubulin mixtures has been attributed to lipid-protein interactions (Caron, J. M., and R. D. Berlin, 1979, J. Cell Biol. 81:665-671). We show that this stacking is produced by the phosphotungstic acid used for staining, independent of the presence of tubulin in the sample. The morphology of negatively stained single bilayer vesicles obtained from dimyristoyl phosphatidylcholine or egg lecithin is specifically dependent upon the choice of metal stain. Uranyl oxalate maintains the appearance of unilamellar vesicles. After staining with sodium tungstate, the lipids form a network of multilayered lamellae with a periodicity of approximately 55 A. Phosphotungstic acid produces stacks of flattened vesicles with a period of approximately 115 A as well as broader multilamellar structures having a 55 A repeat. The stain-determined morphology is not markedly altered by sample concentration, incubation time, or temperature, or by the presence of tubulin.