Objectives: Potassium channels are expressed ubiquitously in prokaryotic and eukaryotic cells, where one of their key functions is to set the cell membrane potential. Today, more than 70 K⁺ channel genes are known. The diversity of K⁺ channels is further increased by the fact that different K⁺ channel family members may assemble to form heterotetramers. We now advanced a method based on fluorescence microscopy for determining the subunit composition of a tetrameric K⁺ channel. Methods: We generated artificial "heteromers" of the K⁺ channel hK_Ca3.1 by coexpressing two differently tagged hK_Ca3.1 channel constructs containing either an intracellular V5- or an extracellular HA-epitope. hK_Ca3.1 channel subunits were detected in the plasma membrane of MDCK-F cells by labeling the intra- and extracellular epitopes with differently colored quantum dots (QDs). Results: As previously shown for the extracellular part of hK_Ca3.1 channels, the intracellular domain of this channel can also bind only one QD label at a time. When both channel subunits were coexpressed, 27.5 ± 1.8% and 24.9 ± 2.1% consisted of homotetramers formed of HA- and V5-tagged subunits, respectively. 47.6±3.2% of the channels were heteromeric and composed of both subunits. Conclusion: The frequency distribution of HA- and V5-tagged homo- and heteromeric hK_Ca3.1 channels is reminiscent of the binomial distribution (a + b)² = a² + 2ab + b². Along these lines our findings are consistent with the notion that hK_Ca3.1 channels are assembled from two homomeric dimers and not randomly from four independent subunits.