The temperature dependence of psychrophilic and mesophilic (R)-3-hydroxybutyrate dehydrogenase steady-state rates yields nonlinear and linear Eyring plots, respectively. Solvent viscosity effects and multiple- and single-turnover pre-steady-state kinetics demonstrate that while product release is rate-limiting at high temperatures for the psychrophilic enzyme, either interconversion between enzyme-substrates and enzyme-products complexes or a step prior to it limits the rate at low temperature. Unexpectedly, a similar change in rate-limiting step is observed with the mesophilic en-zyme, where a step prior to chemistry becomes rate-limiting at low temperature. This observation may have implica-tions for past and future interpretation of temperature-rate profiles.