Gamma-secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimer's disease-associated beta-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human gamma-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of gamma-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of approximately 500 kDa. Purified gamma-secretase was capable of producing the major amyloid-beta peptide (Abeta) species, such as Abeta40 and Abeta42, from a recombinant APP substrate in physiological ratios. Abeta generation could be modulated by pharmacological gamma-secretase modulators. Moreover, the Abeta42/Abeta40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimer's disease mutant. Tandem mass spectrometry analysis revealed the consistent coisolation of several proteins with the known gamma-secretase core subunits. Among these were the previously described gamma-secretase interactors CD147 and TMP21 as well as other known interactors of these. Interestingly, the Niemann-Pick type C1 protein, a cholesterol transporter previously implicated in gamma-secretase-mediated processing of APP, was identified as a major copurifying protein. Affinity capture experiments using a biotinylated transition-state analogue inhibitor of gamma-secretase showed that these proteins are absent from active gamma-secretase complexes. Taken together, we provide an effective procedure for isolating endogenous gamma-secretase in considerably high grade, thus aiding further characterization of this pivotal enzyme. In addition, we provide evidence that the copurifying proteins identified are unlikely to be part of the active gamma-secretase enzyme.