We describe a rapid fluorescence technique for bacterial "fingerprinting," based on the differences in enzyme content and activity of various bacteria. Bacterial cells are incubated with a mixture of carefully chosen fluorogenic enzyme substrates to produce fluorophores with unique emission and excitation properties. The resulting two-dimensional fluorescence spectrum of the product mixture produces a pattern characteristic of the bacterium. A Fourier-transform-based pattern recognition algorithm is used for spectral matching and for differentiating visually similar spectra. This sensitive technique is potentially applicable to differentiation and identification of bacteria in clinical laboratories.