The effects of temperature on the operation of two ion-coupled cotransporters of the SLC6A family, namely rat GAT1 (SLC6A1) and KAAT1 (SLC6A19) from Manduca sexta, have been studied by electrophysiological means in Xenopus laevis oocytes expressing these proteins. The maximal transport-associated current (I(max)) and the apparent substrate affinity (K(05)) were measured. In addition to the expected increase in transport rate (Q(10) = 3-6), both transporters showed greater K(05) values (i.e., a decrease in apparent affinity) at higher temperatures. The transport efficiency, estimated as I(max)/K(05), increased at negative potentials in both transporters, but did not show statistically significant differences with temperature. The observation that the apparent substrate affinity is inversely related to the transport rate suggests a kinetic regulation of this parameter. Furthermore, the present results indicate that the affinities estimated at room temperature for mammalian cotransporters may not be simply extrapolated to their physiological operating conditions.