There is a marked hysteresis between the heating and cooling polymorphic phase transition of anhydrous cholesterol. At a scan rate of 0.05 degrees C/min the difference in transition temperatures between heating and cooling scans is approximately 10 degrees C. This phenomenon also occurs with mixtures of cholesterol with phosphatidylserine and can result in an underestimation of the amount of crystalline cholesterol in a sample that has not been cooled sufficiently. With 1-palmitoyl-2-oleoyl phosphatidylserine and 1-stearoyl-2-oleoyl phosphatidylserine the cholesterol crystallites form while the lipid remains in the L(alpha) phase. Sonication of dimyristoyl phosphatidylserine with a 0.4 mol fraction cholesterol results in the loss of cholesterol crystallite diffraction, but only a partial loss of the polymorphic transition detected by calorimetry. We therefore conclude that the thermal history of the sample can have profound effects on the appearance of the polymorphic phase transition of cholesterol by differential scanning calorimetry. Depending on the morphology of the vesicles, diffraction methods may underevaluate the amount of cholesterol crystallites present.