Membrane dipeptidase (EC 3.4.13.19) is a widely distributed mammalian cell surface enzyme involved in the metabolism of glutathione, leukotriene D4, and certain beta-lactam antibiotics. In this study we have developed a sensitive and rapid assay for membrane dipeptidase based on the fluorometric detection of the D-Phe released from the model substrate Gly-D-Phe. The released D-Phe is first acted on by D-amino acid oxidase in the presence of flavin adenine dinucleotide. The resulting hydrogen peroxide is then metabolized by peroxidase in the presence of the acceptor substrate p-hydroxyphenylacetic acid which is converted to a highly fluorescent compound. The assay configuration is sensitive down to 0.1 nmol D-Phe and can accurately measure membrane dipeptidase activity even in the presence of large amounts of contaminating protein. The membrane dipeptidase assay and the subsequent fluorometric detection of the released D-Phe can be performed in microtiter plates, thus taking less than 1 h to process 96 samples. This sensitive and rapid assay will be useful for the routine measurement of membrane dipeptidase activity in a number of different applications.