The reactions of the Escherichia coli D-galactosebinding protein with the ligands D-galactose and Dglucose have been examined by stopped flow rapid mixing techniques. The change in tryptophan fluorescence that occurs with galactose binding is described by a rapid, second order process; no additional phases of fluorescence change were observed. The resultant time courses were fitted to a simple equilibrium model and the rate constants for galactose binding were calculated to be k,, = 3.3 x lo’ M“ s-l and koa = 4.6 s-l in 10 nm Tris-HC1, pH 7.4 at 20°C. In addition, the dissociation rate constants for glucose (1.4 s-’) and galactose (4.5 s-’) were determined directly from displacement reactions at high concentrations of the complimentary sugar. In both cases, the time courses were exponential in form and consistent with the first order release of sugar from a single binding site. We have also utilized a novel kinetic approach to determine the association rate constant for glucose binding (3 to 4 % lo7 M-’ s-’). For all of these experiments, the data are consistent with a simple mechanism of sugar binding to a single site on the galactose-binding protein. We have also established that purified galactose-binding protein contains an approximately stoichiometric amount of bound glucose which can be readily removed by dialysis against 2 M guanidine HC1. The presence of bound glucose may account for the heterogeneous galactose equilibrium binding curves which have been reported in the past by other investigators.