The human KCNK4 gene encodes several transcripts that generate two-pore K+ channel subunits. We describe the identification and cloning of two transcripts of this gene: human KT4.1a (HKT4.1a) and HKT4.1b. They encode proteins of 393 and 419 amino acids, respectively. HKT4.1a and mouse TRAAK (mTRAAK) are 83% identical, polymerase chain reaction (PCR) experiments performed with rat and human samples as well as the comparison of the HKT4.1 and mTRAAK UTRs strongly suggest that both the human and mouse cDNAs are products of ortholog genes. In contrast to the reported exclusive expression mTRAAK in the nervous system, human and rat KCNK4 gene products are expressed widely in several tissues. Northern blot analysis revealed the presence of three bands of 1.9, 3.0, and 4.8 kb in human, while in rat four bands of 1.8, 3.6, 5.2 and 8.6 kb were observed. Human KCNK4 transcripts were expressed mainly in the heart and brain but also in the liver, skeletal muscle, kidney and pancreas. In rat, the transcripts were strongly expressed in the brain but were also detected in the lung, kidney, liver, spleen, skeletal muscle, testes and at lower levels in the heart. Expression of HKT4.1b in Xenopus oocytes drives the resting potential close to the potassium equilibrium voltage. The expressed channels are not gated by voltage and are permanently open. The channels are not blocked by the classical K+ channel blockers TEA, 4-AP, Cs+, Ba++, quinine or quinidine. Analysis of genomic sequences reveals that seven exons participate to produce HKT4.1a and 11 exons to produce HKT4.1b cDNAs. The KCNK4 gene maps to chromosome 11q13.