Glucose transport in the rat erythrocyte is subject to feedback regulation by sugar metabolism at high but not at low temperatures [Abumrad et al. (1988) Biochim. Biophys. Acta 938, 222-230]. This indicates that temperature, which is known to alter membrane fluidity, also alters sensitivity of transport to regulation. In the present work, we have investigated a possible correlation between the effects of temperature on rate-limiting steps of glucose transport and on membrane fluidity. The dependences of methylglucose efflux and influx on cis and trans methylglucose concentrations were studied at temperatures between 17 and 37 °C. Membrane fluidity was monitored over the same temperature range by using electron paramagnetic resonance spectroscopy. External sugar did not affect efflux, and the Km and Vmax of sugar exit were respectively the same as the Km and Vmax of equilibrium exchange. These Km's were relatively temperature independent, but the Vmax's increased sharply with temperature. The Km and Vmax of methylglucose entry were respectively much lower than the Km and Vmax of exit and exchange. Consistent with the above, intracellular sugar greatly enhanced sugar influx, and did so by increasing the influx Vmax without affecting the influx Km. Both lines of evidence indicated that the conformational change of the empty sugar-binding site from in-facing to out-facing orientation is the rate-limiting step of sugar entry into the rat erythrocyte. This was the case at all temperatures; however, the discrepancies of coefficients declined significantly with increasing temperature. The temperature dependence of the slowest step (change from in- to out-facing empty carrier) was evaluated. An Arrhenius plot showed that this step had a much greater activation energy below 23 °C than above: 48.5 ± 6.0 kcal/mol compared to 24.1 ± 1.8 kcal/mol. The temperature dependence of membrane fluidity, monitored by electron paramagnetic resonance spectroscopy of the probe 5-nitroxylstearate, also exhibited a transition near 23 °C. Our data indicate that membrane fluidity may be a factor influencing ease of carrier conformation change.