The measurement of reliable Cu(I) protein binding affinities requires competing reference ligands with similar binding strengths; however, the literature on such reference ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-soluble monovalent copper ligands, MCL-1, MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu(I) in aqueous solution. X-ray structural data, electrochemical measurements, and an extensive network of equilibrium titrations showed that all three ligands form discrete Cu(I) complexes with 1:1 stoichiometry and are capable of buffering Cu(I) concentrations between 10(-10) and 10(-7) M. As most Cu(I) protein affinities have been obtained from competition experiments with bathocuproine disulfonate or 2,2'-bicinchoninic acid, we further calibrated their Cu(I) stability constants against the MCL series. To demonstrate the application of these reagents, we determined the Cu(I) binding affinity of CusF (log K = 14.3 +/- 0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in Escherichia coli. Altogether, this interconnected set of affinity standards establishes a reliable foundation that will facilitate the precise determination of Cu(I) binding affinities of proteins and small-molecule ligands.