Abstract: Both apolipoprotein E (apoE) and the low-density lipoprotein (LDL) receptor are present in brain; however, little is known regarding the function of these proteins in brain, in particular with respect to brain cholesterol. The role of apoE and the LDL receptor in modulating the transbilayer or asymmetric distribution of cholesterol in the exofacial and cytofacial leaflets of synaptic plasma membranes (SPMs) was examined in mutant mice deficient in apoE, the LDL receptor, or both proteins by using the fluorescent sterol dehydroergosterol and fluorescent quenching procedures. Fluidity of the exofacial and cytofacial leaflets was also measured. Cholesterol asymmetry of SPMs was altered in the mutant mice, with the largest effect observed in the LDL receptor-deficient mice. There was an approximately twofold increase in the percent distribution of cholesterol in the exofacial leaflet of the LDL receptor-deficient mice (32%) compared with C57BL/6J mice (15%). Mice deficient in apoE or both proteins also showed a significantly higher percent distribution of cholesterol (23 and 26%, respectively) in the exofacial leaflet compared with the C57BL/6J mice. Although the percent distribution of cholesterol was highest in the exofacial leaflet of the LDL receptor-deficient mice, fluidity of the exofacial leaflet of that group was significantly lower. However, the cholesterol-to-phospholipid ratio of SPMs of the LDL receptor-deficient mice was significantly lower, and this difference was largely the result of a significant increase in the total amount of SPM phospholipid. This study demonstrates for the first time that SPM lipid structure is altered in mice deficient in apoE or the LDL receptor. Although the mechanism that maintains the asymmetric distribution of cholesterol in plasma membranes is not well understood, data of the present experiments indicate that both apoE and the LDL receptor are involved in maintaining the transbilayer distribution of cholesterol.