Usually K2P background K+ channels assemble as homodimers. Coexpression of TREK-1 and TREK-2 (TWIK-related K+ channel 1 and 2) K2P channel subunits in native cells raised the question whether they can form heterodimers, thus increasing the diversity of background K+ currents, leading to delicate regulation of the resting membrane potential and cell excitability. TREK-1/TREK-2 heterodimerization was enforced artificially by covalently linking the two subunits. Biophysical and pharmacological properties of this model construct were tested and compared with those of the homodimers. Then TREK-1 and TREK-2 were coexpressed and heterodimerization was evaluted by analyzing the appearance of properties characteristic of the tandem construct. Finally, TREK-1/TREK-2 heterodimerization was detected in a native tissue. Channels were expressed in Xenopus laevis oocytes. Currents were measured in whole cells and in excised membrane patches. Dorsal root ganglion (DRG) neurons were isolated from mice and used for single channel recording. Results are given as mean±SEM, statistical analysis was done using ANOVA. Extracellular acidification (to pH 6.5) inhibited TREK-1 (by 49.7±4.3%, n=10 oocytes) and the tandem channel (by 32.7±1.4%, n=8), while TREK-2 was activated (243.3±17.5% of the control, n=6). TREK-1 was insensitive to 30 µM ruthenium red (RR) (9.6±2.3% inhibition, n=10), while TREK-2 was potently inhibited (85.9±2.3%, n=6). The tandem was moderately sensitive to RR (49.4±4.3% inhibition, n=8). Coexpression of TREK-1 and TREK-2 subunits resulted in a current with pH and RR sensitivity suggesting spontaneous heterodimeric assembly. The tandem can also be distinguished from the homodimers (TREK-1: 39.2±2.7 pS, n=6; TREK-2:148.8±4.2 pS, n=7 patches) on the basis of its single channel conductance (83.5±5.0 pS, n=8, p<0.01). Coexpression of TREK-1 and TREK-2 resulted in channels with conductances characteristic of the heterodimer. The effects of RR and the selective TREK-1 blocker spadin (described as an endogenous antidepressant) were examined in outside-out oocyte membrane patches. The effect of RR on TREK channels was confirmed on the single channel level. TREK-1 was inhibited by 1 µM spadin (by 60±11.2%, n=4), while TREK-2 was resistant (101±14.4% of control, n=4). The tandem was also sensitive to spadin (85±12% inhibition, n=5). Single channel currents were recorded in outside-out patches excised from DRG neurons. Nine out of 21 channels were sensitive to both RR and spadin, which is characteristic of the heterodimer. TREK-1 and TREK-2 subunits form functional heterodimers when coexpressed in Xenopus oocytes and in DRG neurons. The heterodimer is inhibited by extracellular acidification and spadin. We propose that the heterodimer may play a role in the sensing of changes in extracellular pH and mediate the antidepressant effect of spadin.