Dextran and methacrylated dextrans (dex-MA) were degraded with dextranase, and the formed degradation products were characterized by electrospray mass spectrometry. It was shown that the main degradation product was isomaltose for both dextran and dex-MA. In degraded dex-MA, the main methacrylated product was isomaltotriose. The relative contribution of oligosaccharides with a higher molecular weight (up to isomaltohexaose) and of multiply methacrylated oligosaccharides increased with the degree of substitution of dex-MA. Enzyme kinetics with a three-substrate model showed that the Michaelis-Menten constant for the monomethacrylated substrate was smaller than for the unsubstituted substrate, whereas the Michaelis-Menten constant for multiply methacrylated susbtrates was higher. This indicates a favorable interaction of one methacrylate group with a hydrophobic binding subsite in the enzyme. The maximum degradation rate, however, was substantially lower for the substituted substrates than for the native substrate. From these results. it is concluded that the enzyme hydrolyzes a glycosidic bond between a methacrylated glucopyranose residue and an unsubstituted one in the dex-MA chain. This hypothesis is further supported with electrospray mass spectrometry because of both the presence of an ion formed by fragmentation at the nonreducing end of an oligosaccharide and the absence of oligosaccharides in which the number of methacrylates equals or exceeds the number of glucopyranose residues.