The level of a normally minor component of the membrane polar lipids of Acholeplasma laidlawii B was significantly increased when the glucose supplement in the growth medium was reduced. Under such glucose-limiting conditions the proportion of this component was found to be dependent upon the fatty acid supplement and could approach 55-60% of the total polar lipids when palmitic acid was used to supplement the growth medium. A number of physical measurements, along with specific chemical and enzymic degradation studies followed by a careful analysis of the degradation products, enabled us to tentatively identify this lipid as a polyprenyl-alpha-D-glucoside with a long-chain fatty acid esterified to the 2-hydroxyl group of the sugar moiety. This lipid exhibited some unusual thermotropic phase properties and our observations suggest that it may not be easily miscible with the other membrane lipid components. The structure and physical properties of this unusual glycolipid also suggest that it may be capable of forming non-bilayer phases under physiologically relevant conditions.