A rapid enzymatic two-step test for the presumptive differentiation of four Candida species commonly occurring in various clinical samples is described. The technique involves membrane filtration of a liquid sample, followed by preincubation of the membrane filter on Sabouraud glucose agar supplemented with ticarcillin-clavulanic acid to yield microcolonies. In a separate assay step, parts of the filter are placed on absorbent pads impregnated with fluorogenic 4-methylumbelliferyl (4-MU) enzyme substrates (4-MU-N-acetyl--D-galactosaminide, 4-MU-phosphate, 4-MU-pyrophosphate, and 4-MU--D-galactoside) in combination with 0.1% digitonin acting as a membrane permeabilizer. The membrane filter in contact with the assay medium is incubated to allow cleavage of the enzyme substrate, resulting in fluorescent microcolonies under long-wavelength UV light. This approach, tested on 301 clinical samples, is able to presumptively differentiate C. albicans, C. glabrata, C. krusei, and C. tropicalis and to distinguish them from other Candida spp. in about 9 to 11 h. Overall agreement with the conventional methods of 94.4% (one Candida species present in the sample) to 83.8% (multiple Candida spp. present) was obtained. The false-negative rates with reference to identification by traditional methods were 1.3% (single species) and 3.8% (multiple species).