It has been proposed that the lower levels of exogenous cholesterol incorporation into the membranes of the sterol-non-requiring as compared to the sterol-requiring mycoplasmas may be due to the much higher glycolipid content of the former and to the reduced ability of glycolipids, as opposed to phospholipids, to incorporate sterols [Efrati et al. (1986) Arch. Biochem. Biophys. 248, 282-288]. In order to test this hypothesis, we have investigated the interaction of cholesterol with the major membrane glyco- and phospholipids of the sterol-non-requiring mycoplasma Acholeplasma laidlawii B, utilizing elaidic acid-homogenous membranes in order to obviate any differences in the nature of cholesterol-lipid interactions due to variations in the fatty acid composition of the different membrane components. Specifically, we have studied the effect of increasing quantities of cholesterol on the thermotropic phase behavior of aqueous dispersions of phosphatidylglycerol, diglucosyl diacylglycerol, and monoglucosyl diacylglycerol, as well as the total membrane polar lipids of this organism, using high-sensitivity differential scanning calorimetry. We find that cholesterol is highly miscible in both the lamellar gel and liquid-crystalline states of phosphatidylglycerol but exhibits limited miscibility in the two neutral glycolipids, particularly in their lamellar gel and crystalline states. We also demonstrate that cholesterol has a limited miscibility in both the lamellar gel and liquid-crystalline states of bilayers composed of the total A. laidlawii B membrane polar lipids. These results demonstrate that the nature of cholesterol-lipid interactions depends markedly on the structure of the glycerolipid polar headgroup and suggests that the incorporation of lower levels of cholesterol into the membranes of the sterol-non-requiring mycoplasmas may indeed be due, at least in part, to their high glycolipid contents. We also show that cholesterol stabilizes the lamellar liquid-crystalline phase of the monoglucosyl diacylglycerol relative to the inverted hexagonal phase at all sterol concentrations, in contrast to the effects of cholesterol on dielaidoylphosphatidylethanolamine, which destabilizes the lamellar liquid-crystalline phase at low concentrations.