Background: Formation of deposits of the insoluble amyloid β-peptide is believed to be causally related with neurodegeneration in Alzheimer disease (AD). The β-peptide originates from a larger amyloid precursor protein (APP) by the action of proteolytic enzymes. The first proteolytic event leading to amyloid formation is the cleavage of APP by the membrane-bound aspartyl protease BACE-1, also known as memapsin-2. Inhibition of BACE-1 is thought to be a therapeutic approach to AD. Measuring BACE-1 activity in biological samples would be useful to elucidate the mechanism of AD and for development of AD drugs. Methods: We developed a sensitive and specific activity assay for BACE-1. The assay is based on a genetically engineered proenzyme that is specifically activated by BACE-1. The resulting active enzyme is measured with a chromogenic substrate. The use of 2 coupled reactions produces a detection limit as low as 0.4 pmol/L. Results: The assay detected BACE-1 activity in extracts of human brain tissue as well as, unexpectedly, in human cerebrospinal fluid (CSF). Gel electrophoresis and Western blotting identified the BACE-1 present in CSF as a truncated soluble form of the originally membrane-bound BACE-1. Conclusion: Detection of the soluble form of BACE-1 in CSF, a relatively easily accessible biological fluid, may be useful for monitoring the effects of drug candidates in vivo and may have diagnostic or prognostic applications. © 2006 American Association for Clinical Chemistry. Chemicals / CAS: amyloid beta protein, 109770-29-8; aspartic proteinase, 78169-47-8; cathepsin D, 9025-26-7; renin, 61506-93-2, 9015-94-5; Amyloid Precursor Protein Secretases, EC 3.4.-; Aspartic Endopeptidases, EC 3.4.23.-; BACE1 protein, human, EC 3.4.23.46; CASP3 protein, human, EC 3.4.22.-; Caspase 3, EC 3.4.22.-; Caspases, EC 3.4.22.-; Endopeptidases, EC 3.4.-; Protein Precursors; Tissue Extracts