Commonly used fluorogenic substrate analogues for the detection of protease activity contain two enzyme-cleavable bonds conjugated to the fluorophore. Enzymatic cleavage follows a two-step reaction with a monoamide intermediate. This intermediate shows fluorescence at the same wavelength as the final product complicating the kinetic analysis of fluorescence-based assays. Fluorogenic substrate analogues for a-chymotrypsin with one cleavable peptide bond have been prepared from morpholinecarbonyl-Rhodamine 110 (MC-Rh 110). A comparison of their kinetic properties with the corresponding (peptide)(2)-Rh110 derivatives revealed that these frequently used double-substituted substrate analogues yield only apparent Km and km, values that are quite different from the kinetic parameters obtained from the monosubstituted MC-Rh110 based substrate analogues. Although both the monoamide intermediate and MC-Rh110 are monosubstituted Rhodarnine 110 derivatives, they show different spectroscopic properties. The data from the spectroscopic analysis clearly show that these properties are directly related to the electron structure of the fluorophore and not to the previously proposed equilibrium between the lactone form and the open ionic form of the fluorophore. This knowledge about the determinants of the spectroscopic properties of monosubstituted Rhodamine 110 introduces a way for a more systematic development of new fluorogenic protease substrate analogues.