Apolipoprotein (apo) E4 is a risk factor for heart disease, Alzheimer's disease, and other forms of neurodegeneration, but the underlying mechanisms are unknown. Domain interaction, a structural property that distinguishes apoE4 from apoE2 and apoE3, results in more rapid turnover and lower plasma levels of apoE4. To determine whether domain interaction affects brain apoE levels, we analyzed brain homogenates from human apoE3 and apoE4 knock-in mice, wild-type mice, and Arg-61 apoE mice, in which domain interaction was introduced by gene targeting. As determined on Western blots, the hemibrain, cortex, hippocampus, and cerebellum of knock-in mice had 30-40% lower levels of apoE4 than apoE3, and Arg-61 mice had 25-50% lower apoE levels than wild-type mice. In the CSF, Arg-61 apoE level was 40% lower than the wild-type level. Arg-61 apoE mRNA levels were similar to or slightly higher than wild-type apoE mRNA levels. Thus, the lower Arg-61 apoE levels were not attributable to decreased mRNA levels. In culture medium from heterozygous Arg-61/wild-type and apoE4/apoE3 primary astrocytes, Arg-61 apoE and apoE4 levels were lower than wild-type apoE and apoE3, respectively, suggesting that primary astrocytes secrete lower amounts of Arg-61 apoE and apoE4. These results demonstrate that domain interaction is responsible for the lower levels of both human apoE4 and mouse Arg-61 apoE in mouse brain. Cells may recognize apoE4 and Arg-61 apoE as misfolded proteins and target them for degradation or accumulation. Thus, degradation/accumulation or lower levels of apoE4 may contribute to the association of apoE4 with Alzheimer's disease.