The reducing terminal of hyaluronate was labeled with a fluorogenic reagent, 2-aminopyridine. The pyridylaminohyaluronate was incubated with testicular hyaluronidase for 1 h. After incubation, 4 vol of ethanol was added to the incubation mixture, followed by centrifugation. The fluorescence of the supernatant containing the degradation products of hyaluronidase digestion was then determined by fluorospectrophotometry (excitation wavelength, 320 nm; emission wavelength, 400 nm). It was found that the increase of the pyridylamino products was linearly correlated with enzyme concentration (up to 0.1 national formulary unit), incubation time (up to 60 min), and substrate concentration (up to 2.5 microM). The fluorogenic substrate was also applicable for the determination of crude hyaluronidase. This simple, rapid, and sensitive hyaluronidase assay was made possible by the use of pyridylaminohyaluronate as a substrate.