TREK-1, TREK-2 and TRAAK are members of the two-pore domain K+ (K2P) channel family and are activated by membrane stretch and free fatty acids. TREK-1 has been shown to be sensitive to temperature in expression systems. We studied the temperature-sensitivity of TREK-2 and TRAAK in COS-7 cells and in neuronal cells. In transfected COS-7 cells, TREK-2 and TRAAK whole-cell currents increased approximately 20-fold as the bath temperature was raised from 24 degrees C to 42 degrees C. Similarly, in cell-attached patches of COS-7 cells, channel activity was very low, but increased progressively as the bath temperature was raised from 24 degrees C to 42 degrees C. The thresholds for activation of TREK-2 and TRAAK were approximately 25 degrees C and approximately 31 degrees C, respectively. Other K2P channels such as TASK-3 and TRESK-2 were not significantly affected by an increase in temperature from 24 degrees C to 37 degrees C. When the C-terminus of TREK-2 was replaced with that of TASK-3, its sensitivity to free fatty acids and protons was abolished, but the mutant could still be activated by heat. At 37 degrees C, TREK-1, TREK-2 and TRAAK were sensitive to arachidonic acid, pH and membrane stretch in both cell-attached and inside-out patches. In cerebellar granule and dorsal root ganglion neurones, TREK-1, TREK-2 and TRAAK were generally inactive in the cell-attached state at 24 degrees C, but became very active at 37 degrees C. In cell-attached patches of ventricular myocytes, TREK-1 was also normally closed at 24 degrees C, but was active at 37 degrees C. These results show that TREK-2 and TRAAK are also temperature-sensitive channels, are active at physiological body temperature, and therefore would contribute to the background K+ conductance and regulate cell excitability in response to various physical and chemical stimuli.