The Alzheimer's amyloid protein (A beta) is released hom the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of A beta producing a large secreted derivative (sAPP beta) and an A beta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 releases the secreted derivative sAPP alpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of A beta 40 and A beta 42 in the medium but had no effect on sAPP alpha, A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of A beta 40, A beta 42,and sAPP beta than its parental line (GPI+), When this parental line was treated with PI-PLC, A beta 40, A beta 42, and sAPP beta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and A beta secretion in mammalian cells, The cell-surface GPI-anchored protein(s) involved in A beta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.