Several sensitive substrates for porcine pancreatic elastase, chymotrypsin, and trypsin were prepared that utilize the permanently charged, fluorogenic cation l-methyl-6-aminoquinoline (MAQ+) as the leaving group. Kinetic rates for the hydrolysis of substrates were determined fluorimetrically and compared with analogues having 6-aminoquinoline (6-AQ) as an uncharged leaving group. It was found that substrates containing the quatemized leaving group generally have a higher kcat/Km ratio. An exception to this trend was noted with a trypsin substrate, Bz-DL-Arg-MAQ+. During the course of this investigation, several significant advantages of the MAQ+ ion as a fluorogenic leaving group in protease substrates were found: (a) its appearance can be measured fluorimetrically using wavelengths of light that result in its maximal fluorescence, while under these conditions, the unhydrolyzed substrate is essentially nonfluorescent, (b) it confers a high degree of water solubility to hydrophobic peptides, thereby eliminating the need for organic cosolvents to dissolve substrates, and (c) quatemized substrates can be prepared readily and in good yield from the corresponding 6-(peptidylamido)quinolines. These positively charged synthetic fluorogenic substrates are, therefore, useful probes for investigating the steric and electronic properties of the active-site environment of proteolytic enzymes.