To obtain an ethanolamine plasmalogen (PlsEtn)-hydrolyzing enzyme and to develop an assay that would help determine PlsEtn concentrations in human serum as an indicator of Alzheimer-type dementia and of arteriosclerosis. Phospholipase A(1)s, SaPLA1 and SvPLA1 from, respectively, Streptomyces albidoflavus NA297 and S. avermitilis JCM5070-but not phospholipase B from Streptomyces sp. NA684, PLA2-Nagase from S. avermitilis, PLA(2)IIL from S. violaceoruber nor LIPOMOD 699L (porcine phospholipase)-hydrolyzed choline plasmalogen (PlsCho) and PlsEtn (PlsCho preferred over PlsEtn). Using a combination of SaPLA1, lysoplasmalogen-specific phospholipase D (LyPls-PLD), with amine oxidase, an end-point assay was developed for measuring serum PlsEtn concentration. The standard curve, generated using various amounts of PlsEtn in this assay, was linear between 0 and 0.2 mM. PlsEtn concentrations in forty-seven serum samples, determined independently by this enzyme-based assay and I-125-HPLC method, exhibited a linear relationship, indicating that the assay is suitable for fast and accurate measurement of serum PlsEtn concentration. An assay, developed using SaPLA1, LyPls-PLD, and AOX, selectively measured PlsEtn levels in blood samples. This assay could be a useful diagnostic tool for early stage detection of diseases such as Alzheimer-type dementia and arteriosclerosis.