A series of 6-substituted methyl lactoside derivatives together with methyl allolactoside and (6S)-methyl [6-2H]lactoside have been synthesized and characterized by NMR spectroscopy. All compounds were tested as substrates for the enzyme beta-galactosidase from E. coli using progress curve kinetic methology both in single-substrate and competition experiments. The results show that the hydrolysis of methyl lactoside to a large extent takes place through an intramolecular transglycosidation reaction via allolactoside. Furthermore, methyl 6-amino-6-deoxy-D-glucopyranoside proved to be an ihibitor for the enzymatic hydrolysis.