The ultrastructure of aggregates formed by mixtures of pig erythrocyte lecithin, cholesterol and globoside in aqueous systems was studied by electron microscopy and X-ray diffraction. Globoside and lecithin in up to equimolar amounts formed a lamellar mesophase, although the structure of the lamellae was perturbed. Mixtures containing excess globoside formed complex tubular or reticular aggregates. Cholesterol appeared to promote mixing of lecithin and globoside. The flexibility gradient of the hydrocarbon (hc) region of the lipid bilayers was studied using electron spin resonance (esr) spectroscopy of various nitroxide-labelled stearic acid probes. Globoside in equimolar amounts greatly perturbed the order parameters of lecithin bilayers, reducing the fluidity of the hc region and flattening the flexibility gradient near the polar (p) surface. The effect of globoside on lecithin–cholesterol bilayers was not so pronounced, since the latter was already more ordered than lecithin bilayers. A phase transition of pure globoside at 55 °C, involving 'melting' of the hc chains was also detected using X-ray and esr spectroscopic techniques. The interbilayer spacing, dw, of equimolar lecithin–globoside lamellar phase increased by 42% from that of lecithin bilayers, indicating that the glycolipid p group may increase the net repulsive force between bilayers, as was previously predicted theoretically.