A total of 712 strains representing 47 taxa of the family Enterobacteriaceae were tested for the ability to hydrolyze 14 4-methylumbelliferyl (4-MU)-linked substrates within 3 h of incubation. In addition to the well-known differentiation potential of the hydrolysis of 4-MU-beta-D-galactopyranoside, 4-MU-beta-D-glucuronide, and 4-MU-beta-D-xylopyranoside, the hydrolysis of some other fluorogenic substrates (e.g., 4-MU-beta-D-fucopyranoside, 4-MU-N-acetyl-beta-D-galactosaminide, and 4-MU-alpha-D-galactopyranoside) can also be used for species differentiation within the family Enterobacteriaceae.