TREK-2 expressed in mammalian cells exhibits small (∼52 pS) and large (∼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 (Δ1–36) of the N-terminus had no effect. However, deletion of most of the N-terminus (Δ1–66) resulted in the appearance of only the large-conductance channel (∼220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants Δ1–44 and Δ1–54 produced ∼90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at ∼54 kDa and ∼60 kDa from cells expressing wild-type TREK-2 that has three potential translation initiation sites (designated M1M2M3) within the N-terminus. Mutation of the second and third initiation sites from Met to Leu (M1L2L3) produced only the ∼60 kDa isoform and the small-conductance channel (∼52 pS). Mutants designed to produce translation from the second (M2L3) or third (M3) initiation site produced the ∼54 kDa isoform, and the large conductance channel (∼185–224 pS). M1L2L3, M2L3 and M3 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. PNa/PK values were also similar for M1L2L3 (∼0.02), M2L3 (∼0.02) and M3 (∼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.