The accumulation and deposition of fibrillar A beta is thought to be the primary cause of Alzheimer's disease. A beta is derived from Alzheimer amyloid precursor protein (APP) by sequential proteolytic cleavage involving beta- and gamma-secretase. Recently, gamma-secretase was shown to cleave near the cytoplasmic membrane boundary of APP (called the epsilon-cleavage), as well as in the middle of the membrane domain (gamma-cleavage). It has been reported that the C-terminus of A beta is generated via a series of sequential cleavages, epsilon-cleavage followed by gamma-cleavage. However, recent article has reported that gamma- and epsilon-site cleavage are regulated independently. The relationship between gamma-site and epsilon-site cleavage is still unknown. In this study, we analyzed the generation of AICD and A beta in CHO cells expressing APP derivatives. We found that epsilon-site cleavage preferentially occurs alpha-secretase processing product, and that A beta 11-40/42 was generated without gamma-secretase epsilon-site cleavage, indicating that gamma-site cleavage and epsilon-site cleavage were regulated differentially. (c) 2006 Elsevier Inc. All rights reserved.