The kinetics of cellulose binding and hydrolysis by cellulases is not well understood except at steady-state conditions. For use in studies of cellulase pre-steady-state and steady-state kinetics, we have prepared 4-methyl-7-thioumbelliferyl-beta-D-cellobioside (MUS-CB), a ground-state nonhydrolyzable analogue of the fluorescent cellulase substrate 4-methylumbelliferyl-beta-D-cellobioside (MU-CB). MUS-CB is not hydrolyzed by the catalytic domain of cellulase E1 from Acidothermus cellulolyticus under conditions where this enzyme rapidly degrades MU-CB. Thermodynamic parameters describing the steady-state binding of MUS-CB to Thermobifida fusca cellulase Cel6A are similar to those for MU-CB, indicating that MUS-CB can be used in place of MU-CB to study binding events in the Cel6A active-site cleft. In the pre-steady-state, MUS-CB binds to Cel6A by a simple, one-step bimolecular association reaction. It is anticipated that similar thio-containing 4-methylumbelliferyl compounds will have applications in studies of other enzyme systems.