Two-pore Domain Potassium (K2P) Channels are the most recently identified potassium channel class. They are widely expressed in the body – in the central nervous system as well as the periphery, e.g. lung, heart and endocrine cells – with strongly overlapping expression patterns of the 15 K2P members. Their large basal activity hyperpolarizes the resting membrane potential and dampens the excitability of neurons and other cells. K2P channels are modulated by a variety of mechanical and chemical stimuli like stretch, heat, acidification, lipids or anaesthetics, denoting their role in many physiological and pathophysiological processes including sleep, pain or epilepsy. Unlike other K+ channels, they show a particular topology of four transmembrane helices with two pore domains. Therefore, only two subunits assemble as dimers with the typical tetrameric symmetry of the pore. The 15 K2P members are classified into six subfamilies by sequence similarity and most channels form functional homodimers in heterologous expression systems. Only a few heterodimers within the subfamilies have been reported, e.g. THIK-1/THIK-2, TREK-1/TREK-2, TASK-1/TASK-3. Nearly nothing is known about the formation of heterodimeric channels across the K2P subfamilies. We used bimolecular fluorescence complementation (BiFC) to systematically screen for all possible heterodimers. All K2P subunits were cloned into the pBiFC-VN173 and pBiFC-VC155 vectors, producing fusion proteins with half of a split fluorescent protein each. All possible combinations were coexpressed, and fluorescence by recombination of split proteins revealed the formation of several heterodimeric channels also across different subfamilies. We particularly examined the TWIK/TALK combination because of a strong fluorescence in the BiFC assay, and estimated the homodimeric and heterodimeric fractions of the coexpressed BiFC constructs. Co-immunoprecipitation experiments confirmed their existence and they were characterized in electrophysiological measurements. Moreover, fluorescence microscopy showed a change in TWIK-1 localization compared to the wildtype homodimer. Interestingly, TWIK-1 mRNA is ubiquitously expressed, but the homodimeric channel protein is reported to be nonfunctional because of intracellular sequestration. Hence, heterodimerization may be a requirement for functional plasma membrane expression of TWIK-1, and TWIK family members could function as regulator subunits for many K2P channels from other subfamilies, modulating trafficking, plasma membrane expression levels and gating characteristics. The cross-subfamily combination of K2P channel monomers could confer intermediate or even completely new properties to these mixed channels and contribute to the diversity of K+ channels adapted to their specific cellular environment in vivo.