Sixty representatives of selected Mycobacterium and Nocardia species were examined for their ability to cleave 79 fluorogenic synthetic enzyme substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone. The resultant data were analysed using the simple matching coefficient and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. Clusters corresponding to the validly described species Mycobacterium bovis, M. chelonae, M. chitae, M. farcinogenes, M. fortuitum, M. peregrinum, M. senegalense, M. smegmatis, Nocardia asteroides, and N. farcinica were circumscribed at or above the 83% similarity level. Fluorogenic probes prepared from 7-amino-4-methylcoumarin and 4-methylumbelliferone provide a rapid means of detecting taxonomically useful enzymes in small amounts of whole mycobacteria and nocardiae.