The application of a new fluorescence-quenching-resolved spectroscopic method [Wasylewski, Z., Kołoczek, H. and Waśniowska, A. (1988) Eur. J. Biochem. 172, 719-724] for resolving fluorescence emission spectra of a mixture of fluorophores into components is described. Contrary to fluorescence lifetime measurements, in this method the overlapping spectra can be decomposed even if the components have similar or the same fluorescence lifetimes, but differ in bimolecular-rate-quenching constants. Using this technique, we have resolved the emission spectra of a two-component mixture of fluorescein and riboflavin, which have very similar fluorescence lifetimes. To illustrate the utility of this approach in the study of fluorophores in compartmentalized biological systems such as lipid bilayers, we have also used the method to resolve the emission spectra of a two-component mixture of fluorophores commonly used in biological studies which undergo partition between water and a micellar phase.