The subunits of the voltage-gated potassium channels Kv1.1 and Kv1.2 can form both homo- and heterotetrameric channels in cells, the functional properties and localization of which differ significantly. Confocal microscopy based on Förster resonance energy transfer was used to study the formation of Kv1 channels during the co-expression of Kv1.1(S369T) and Kv1.2(S371T) subunits in mouse neuroblastoma cells, which were fused with the fluorescent protein mKate2 and TagCFP, respectively, and had enhanced membrane expression due to mutation. It was found that TagCFP-Kv1.1(S369T) and mKate2-Kv1.2(S371T) effectively formed heterochannels that were localized both in the plasma membrane and in the cytoplasm of cells. In the absence of the S369T mutation, heterochannels did not integrate into the plasma membrane, which indicated the need for auxiliary factors for the transfer of native heterochannels into the membrane. In addition to heterochannels, homotetrameric channels were formed in Neuro-2a cells; however, the efficiency of heterochannel formation was much higher.