Fluorescent labeling of proteins is one of the most popular and applicable methods in molecular diagnostic studies used for quantification assays, as well as for determination of protein–protein or ligand–protein interactions.[1, 2] The imaging of the labeled proteins is of great importance for the understanding of protein function, intra-or intercellular distribution of proteins, and sensing of biological events. Genetically encoded sensors derived from fluorescent proteins (GFP and others) are extremely useful for those studies; however, the large size and spectral properties of fluorescent proteins introduce certain limitations for their applications. In the last decade, researchers successfully imaged proteins in cells by using biarsenical fluorescein (FlAsH-EDT2) developed by RY Tsien and co-workers.[3] This technique requires a short tetracysteine tag (originally employed sequence: CCPGCC) added to the N or C terminus of the protein, to flexible protein loops, or further in vitro or in vivo labeling with biarsenical probes.[4] Those probes, practically nonluminescent, become highly fluorescent upon formation of a complex with tetracysteine motifs. Recent developments include successful application of biarsenical probes with various spectral properties for FRET or for in vivo photocrosslinking.[5–8] The elongation of the tetracysteine tag to three repeating CCPGCC motifs improved ReAsH-EDT2 brightness per peptide both in vitro and in mammalian cells.[9] Despite the successful application of biarsenical probes to label tetracysteine motifs in proteins, they have a number of drawbacks, including a requirement for reduced cysteines, limited penetration through bacterial cell walls, and high background, which results from the binding of biarsenical dyes to the endogenous cysteine-rich proteins or hydrophobic pockets of proteins.[5, 10] One of the major limitations for this fluorescence technique is a tendency of CCPGCC motifs to aggregate through formation of disulfide bridges under slightly oxidizing conditions.[11] To minimize that drawback Wang et al. recently identified alternative amino acid binding motifs for FlAsH-EDT2 modification with a longer spacing between cysteine residues (nine and 15 amino acids) that did not result in loss of fluorescent properties of the complex.[12] Here, we present systematic studies on the interaction of the classical tetracysteine motif CCPGCC with ZnII, one of the most widespread biological elements with high affinity to cysteine-containing proteins, for example, metallothioneins and zinc fingers.[13, 14] ZnII is a catalytic and structural cofactor for thousands of proteins that bind ZnII with high affinity (mostly in the range of nM and pM Kd); however, its tightly controlled fluctuations modulate signaling pathways.[14–16] Extra-and intracellular concentrations of thermodynamically and kinetically mobile ZnII (“free zinc”) vary several orders of magnitude and are also strongly dependent on cellular compartmentalization.[14, 17] Recent studies clearly demonstrate intracellular “free” zinc concentration at a level of hundreds of pM in different types of eukaryotic cells; 400 pM in pancreatic β cells and HEK-293,[18] 784 pM in HT-29 cells,[16] 500 pM in neuroblastoma cells [19] and 100–870 pM in cardiomyocytes.[20, 21] However, typical total zinc concentration of a eukaryotic cell is between approximately 200–300 μM.[16] We report here that the common CCPGCC tetracysteine sequence has high affinity for ZnII with a binding constant similar to the affinity of native zinc proteins. This high affinity causes ZnII transfer from the zinc pool to CCPGCC motifs; this significantly decreases the binding efficiency of this motif to FlAsH-EDT2 and other …