The number of reported cases of chronic arsenic poisoning is on the rise throughout the world, making the study of the long-term effects of arsenic critical. As3+ binds readily to biological thiols, including mammalian metallothionein (MT), which is an ubiquitous sulfur-rich metalloprotein known to coordinate a wide range of metals. The two-domain mammalian protein binds divalent metals (M) into two metal-thiolate clusters with stoichiometries of M3Scys9 (beta) and M4Scys11 (alpha). We report that As3+ binds with stoichiometries of As3Scys9 (beta) and As3Scys11 (alpha) to the recombinant human metallothionein (rhMT) isoform 1a protein. Further, we report the complete kinetic analysis of the saturation reactions of the separate alpha and beta domains of rhMT with As3+. Speciation in the metalation reactions was determined using time- and temperature-resolved electrospray ionization mass spectrometry. The binding reaction of As3+ to the alpha and beta MT domains is shown to be noncooperative and involves three sequential, bimolecular metalation steps. The analyses allow for the first time the complete simulation of the experimental data for the stepwise metalation reaction of MT showing the relative concentrations of the metal-free, apo MT and each of the As-MT intermediate species as a function of time and temperature. At room temperature (298 K) and pH 3.5, the individual rate constants for the first, second, and third As3+ binding to apo-RMT are 5.5, 6.3, and 3.9 M-1 s(-1) and for apo-beta MT the constants are 3.6, 2.0, and 0.6 M-1 s(-1). The activation energy for formation of As-1-H-6-beta MT is 32 kJ mol(-1), for As-2-H-3-beta MT it is 35 kJ mol(-1), for As-3-beta MT it is 29 kJ mol(-1), for As-1-H-8-alpha MT it is 33 kJ mol(-1), for As-2-H-5-alpha MT it is 29 kJ mol(-1), and for As-3-H-2-alpha MT it is 23 kJ mol(-1).