We have designed a nanosensor to study the potential function of metallothionein (MT) in metal transfer and its interactions with redox partners and ligands by attaching two fluorescent probes to recombinant human MT. The specific labeling takes advantage of two different modification reactions. One is based on the fact that recombinant MT has a free N-terminal amino group when produced by the IMPACT T7 expression and purification system, the other on the observation that one human MT isoform (1b) contains an additional cysteine at position 32. It is located in the linker region of the molecule, allowing the introduction of a probe between the two domains. An S32C mutation was introduced into hMT-2. Its thiol reactivity, metal binding capacity, and CD and UV spectra all demonstrate that the additional cysteine contains a free thiol(ate); it perturbs neither the overall structure of the protein nor the formation of the metalthiolate clusters. MT containing only cadmium was labeled stoichiometrically with Alexa 488 succinimidyl ester at the N terminus and with Alexa 546 maleimide at the free thiol group, followed by conversion to MT containing only zinc. Energy transfer between Alexa 488 (donor) and Alexa 546 (acceptor) in double-labeled MT allows the monitoring of metal binding and conformational changes in the N-terminal beta-domain of the protein.