J774 macrophages load with cholesteryl ester (CE) when incubated with acetylated low-density lipoprotein and cholesterol-rich liposomes; the CE accumulates as cytoplasmic inclusions 1-2 micron in diameter. The CE core of the droplet comprises about 90% of its mass; the predominant CE species present are cholesteryl palmitate (CP, 41%) and cholesteryl oleate (CO, 37%). The thermotropic properties of the inclusions, both in intact cells and after isolation, have been characterized by differential scanning calorimetry. On heating, the inclusions exhibit two endothermic transitions at about 41 and 53 degrees C with a total enthalpy of 7.7 +/- 1.2 cal/g of CE. Very similar thermal behavior is exhibited by a binary mixture containing equal weights of CO and CP; this indicates that these two species dominate the phase behavior of CE in J774 inclusions. A phase diagram for the CO/CP system has been generated, and this reflects simple eutectic behavior. The eutectic is 83% w/w CO, and it melts at 49-50 degrees C. Below this temperature, CO and CP form two immiscible crystalline phases due to the very limited ability of the unsaturated oleate and saturated palmitate acyl chains to mix in the crystal phase. On heating a 1/1 w/w CO/CP mixture, an isotropic liquid of eutectic composition forms at 49 degrees C, and the remaining crystalline cholesteryl palmitate melts over the temperature range 50-69 degrees C. The phase diagram indicates that bulk mixtures of CE molecules in J774 inclusions should be crystalline at 37 degrees C, the growth temperature of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)