Previous investigations performed on different isoforms of the oligopeptide transporter PepT1, from two poikilotherms (seabass and zebrafish) and one homeotherm animal (rabbit), showed marked differences in the kinetic behaviour of the proteins. Aim of this work was to characterize the relationship between temperature and the functional properties of PepT1 by electrophysiological methods. Raising the temperature in the range 20 to 30 °C causes an increase in the maximal transport-associated current (Imax ) with a Q10 close to 4. Higher temperatures accelerate the rate of decline of the presteady-state currents observed in the absence of organic substrate. The voltage dependencies of the intramembrane charge movement and of the time constant of decline are both shifted towards more negative potentials by higher temperatures. The shift is due to a stronger action of temperature on the outward rate of charge movement compared to the inward rate, indicating a lower activation energy for the latter process. The activation energy for the complete cycle is similar to that of the inward rate of charge movement. Temperature also affects the binding rate of the substrate: the K0.5 –V curve is shifted to more negative potentials by higher temperatures, resulting in a lower apparent affinity in the physiological range of potentials. The overall efficiency of transport, estimated as the I max /K0.5 ratio, is significantly increased at body temperature.