TREK-2 expressed in mammalian cells exhibits small ( approximately 52 pS) and large ( approximately 220 pS) unitary conductance levels. Here we tested the role of the N-terminus (69 amino acids long) in the control of the unitary conductance, and role of the alternative translation initiation as a mechanism that produces isoforms of TREK-2 that show different conductance levels. Deletion of the first half (Delta1-36) of the N-terminus had no effect. However, deletion of most of the N-terminus (Delta1-66) resulted in the appearance of only the large-conductance channel ( approximately 220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants Delta1-44 and Delta1-54 produced approximately 90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at approximately 54 kDa and approximately 60 kDa from cells expressing wild-type TREK-2 that has three potential translation initiation sites (designated M(1)M(2)M(3)) within the N-terminus. Mutation of the second and third initiation sites from Met to Leu (M(1)L(2)L(3)) produced only the approximately 60 kDa isoform and the small-conductance channel ( approximately 52 pS). Mutants designed to produce translation from the second (M(2)L(3)) or third (M(3)) initiation site produced the approximately 54 kDa isoform, and the large conductance channel ( approximately 185-224 pS). M(1)L(2)L(3), M(2)L(3) and M(3) were relatively selectively permeable to K(+), as judged by the 51-55 mV shifts in reversal potential following a 10-fold change in [K(+)](o). P(Na)/P(K) values were also similar for M(1)L(2)L(3) ( approximately 0.02), M(2)L(3) ( approximately 0.02) and M(3) ( approximately 0.03). Arachidonic acid, proton and membrane stretch activated, whereas dibutyryl-cAMP inhibited all three isoforms of TREK-2, indicating that deletion of the N-terminus does not abolish modulation. These results show that the small and large conductance TREK-2 channels are produced as a result of alternative translation initiation, producing isoforms with long and short N-termini, and that the distal half of the N-terminus controls the unitary conductance.