Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.