The intermediate-conductance calcium-activated potassium channel KCa3.1 promotes calcium-dependent hyperpolarization of the cell membrane. Its malfunction has been observed inautoimmune and oncological diseases. To study this channel and its peptide blockers usingfluorescence analysis, plasmids encoding the α-subunit KCa3.1 fused with the fluorescent proteinmKate2 at the N- or C-terminus were constructed, and the fluorescent ligand ChTx-GFP wasobtained, which is a combination of the peptide blocker charybdotoxin and the green fluorescentprotein. It was found that mKate2 at the N-terminus of the α-subunit blocks the transport of thechannel into the plasma membrane of Neuro-2a cells, while mKate2 at its C-terminus does notinterfere with the efficient accumulation of the channel in the plasma membrane and theformation of a regular tetrameric structure capable of binding peptide blockers. The ligandChTx-GFP binds to the KCa3.1 channel on the membrane at a concentration of 20 nM and canbe used for fluorescent imaging of these channels in mammalian cells.