The rates of the forward and reverse reactions of triosephosphate isomerase catalyzed by the wild-type and by a sluggish mutant enzyme have been studied in the absence and the presence of several viscosogenic agents. For the mutant enzyme, the kcat for which is some 10(3) times less than that for the wild-type enzyme, the value of kcat/Km with glyceraldehyde phosphate as substrate is almost unaffected by the presence of sucrose or glycerol, even though the concentration of the aldehyde form of the substrate is smaller because of hemiacetal formation. [The nature and relative amounts of the various forms of triose phosphate present in solution (free carbonyl forms, hydrates, dimers, hemiacetal adducts) have been evaluated by 31P NMR and are presented in the Appendix.] The viscosogenic agents cause the substrate to bind more tightly to the enzyme, roughly compensating for the lower substrate concentration. With dihydroxyacetone phosphate as substrate, the values of kcat/Km for the mutant enzyme increase with the addition of viscosogenic agent, consistent with the tighter binding of substrate without (in this case) any concomitant loss due to hemiketal formation. These results for the mutant enzyme (known to be limited in rate by an enolization step in the catalytic mechanism) can be used to interpret the behavior of the wild-type enzyme. Plots of the relative values of kcat/Km for catalysis by the wild-type enzyme (normalized with the corresponding data for the mutant enzyme) against the relative viscosity have slopes close to unity, as predicted by the Stokes-Einstein equation for a cleanly diffusive process. In the presence of polymeric viscosogenic additives such as poly(ethylene glycol), polyacrylamide, or ficoll, no effect on kcat/Km is seen for the wild-type enzyme, consistent with the expectation that molecular diffusion rates are unaffected by the macroviscosity and are only slowed by the presence of smaller agents that raise the microviscosity. These results show that the reaction catalyzed by the wild-type triosephosphate isomerase is limited by the rate at which glyceraldehyde phosphate encounters, or departs from, the active site.