Abstract: 4-Methyl-7-(phenylacetylamino)coumarin and 7-(phenylacetylamino)-4-(trifluoromethyl)coumarin (prep. described) were used to assay penicillin amidase. The enzyme solution was added to an ethanolic solution of the substrate (diluted with 50 mM potassium phosphate buffer of pH 8) for fluorimetry at 440 and 495 nm, respectively (with excitation at 365 nm). The calibration graphs were rectilinear in the range 20 to 300 µiu mL-1 for both substrates. The compounds can be used for the detection of intracellular enzyme activity in micro-organisms, by fluorescence microscopy or laser - flow cytometry.