A major histopathological hallmark in Alzheimer's disease consists of the extracellular deposition of the amyloid beta-peptide (A beta) that is proteolytically derived from the beta-amyloid precursor protein (beta APP). An alternative, nonamyloidogenic cleavage, elicited by a protease called alpha-secretase, occurs inside the A beta sequence and gives rise to APP alpha, a major secreted C-terminal-truncated form of beta APP. Here, we demonstrate that human embryonic kidney 293 (HK293) cells contain a chymotryptic-like activity that can be ascribed to the proteasome and that selective inhibitors of this enzyme reduce the phorbol 12,13-dibutyrate-sensitive APP alpha secretion by these cells. Furthermore, we establish that a specific proteasome blocker, lactacystin, also induces increased secretion of A beta peptide in stably transfected HK293 cells overexpressing wild-type beta APP751. Altogether, this study represents the first identification of a proteolytic activity, namely, the proteasome, contributing likely through yet unknown intracellular relays, to the alpha-secretase pathway in human cells.