Cholestrol exchange kinetics between [14C]cholesterol‐labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine‐cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.