X11 and X11-like (X11L) are neuronal adaptor proteins whose association to the cytoplasmic domain of APP suppresses the generation of amyloid ß-protein (Aß) implicated in Alzheimer disease pathogenesis. We previously found that the amyloidogenic, but not amyloidolytic, metabolism of APP was selectively increased in the brain of mutant mice lacking X11L (J. Biol. Chem. [2006] 281, 37853-37860), however, the role of X11 proteins (X11s) in the amyloidogenic metabolism of APP was unclear in vivo. To reveal the actual role of X11s in suppressing amyloidogenic cleavage of APP in vivo, we generated X11 and X11L double-knockout mice and analyzed the metabolism of APP. The mutant mice showed enhanced ß-site cleavage of APP along with increased accumulation of Aß in brain and increased co-localization of APP with BACE. In the brains of mice deficient in both X11 and X11L, the apparent relative subcellular distributions of both mature APP and its ß-carboxyl terminal fragment were shifted toward the detergent-resistant membrane (DRM) fraction, an organelle in which ß-site APP-cleaving enzyme (BACE) is active and both X11 and X11L are not nearly found. These results indicate that X11s associate primarily with APP molecules that are outside of DRM, that the dissociation of APP-X11/X11L complexes leads to entry of APP into DRM, and that cleavage of uncomplexed APP by BACE within DRM is enhanced by X11s deficiency. Present results lead to an idea that the dysfunction of X11L in the interaction with APP may recruit more APP into DRM and increase the generation of Aß even if BACE activity did not increase in brain.