Recently, several mammalian K(+) channel subunits (TWIK, TREK-1, TRAAK, and TASK) possessing four transmembrane segments and two pore-forming domains have been identified. We report the cloning of a new member of this tandem-pore K(+) channel from a rat cerebellum cDNA library. It is a 538-amino acid protein and shares 65% amino acid sequence identity with TREK-1. Therefore, the new clone was named TREK-2. Unlike TREK-1, whose mRNA has been reported to be expressed in many different tissues, TREK-2 mRNA is expressed mainly in the cerebellum, spleen, and testis as judged by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Expression of TREK-2 in COS-7 cells induced a time-independent and non-inactivating K(+)-selective current. TREK-2 was partially blocked (36%) by 2 mm Ba(2+). In symmetrical 150 mm KCl, the single-channel conductances were 110 picosiemens at -40 mV and 68 picosiemens at +40 mV, and the mean open time was 0.9 ms at -40 mV. TREK-2 was activated by membrane stretch or acidic pH. At -40 mm Hg pressure, channel activity increased 10-fold above the basal level. TREK-2 was also activated by arachidonic acid and other naturally occurring unsaturated free fatty acids. These results show that TREK-2 is a new member of the tandem-pore K(+) channel family and belongs to the class of mechanosensitive and fatty acid-stimulated K(+) channels.