We have developed a fluorogenic substrate, ELF-97 beta-D-glucuronide, that provides significant advantages over existing substrates in detecting beta-glucuronidase activity. ELF-97 beta-D-glucuronide allows the detection of enzymatic activity in situ, yielding a hydrolytic product that exhibits maximal fluorescence within the physiological pH range. This substrate yields a hydrolytic product that demonstrates a more than 100 nm Stokes shift, which minimizes interference from autofluorescence in plant tissue. With the commercial enzyme, ELF-97 beta-D-glucuronide can detect less than 2 x 10(-4) U/ml of beta-glucuronidase activity in solution, and 5 x 10(-4) U per lane in polyacrylamide gels. Assays using transgenic Arabidopsis, whole leaf extracts of GUS-positive, but not GUS-negative plans, show significant GUS activity upon incubation with ELF-97 beta-D-glucuronide. Furthermore, the ability of this substrate to form insoluble precipitates at the sites of enzymatic activity makes it suitable for in situ localization of GUS activity in tissue samples of higher plants.