Mammalian metallothioneins bind up to 7 Zn2+ in two distinct domains: an N-terminal β-domain that binds 3 Zn2+, and a C-terminal α-domain that binds 4 Zn2+. Domain specificity has been invoked in the metalation mechanism with cluster formation and bridging of the 20 Cys taking place prior to saturation with 7 Zn2+. We report a novel experiment that examines Zn2+ metalation by exploiting the expected decrease in KF at the onset of clustering using ESI-MS. The relative Zn-binding affinities of the 7 total sites in the - and -fragments were determined through direct competition for added Zn2+. The KF's for each Zn2+ are expected to diminish as a function of the remaining available sites and the onset of clustering. Analysis shows that Zn2+ binds to -rhMT with a greater affinity than -rhMT. The incremental distribution of Zn2+ between the competing fragments and apo-βα-rhMT (essentially 3 + 4 sites competing with 7 sites) identifies the exact point where clustering begins in the full protein. During the titration with Zn2+, the ESI-MS data show that several metalated species coexist until such time as the fully saturated proteins are formed. This means that all 20 Cys of apo-βα-rhMT are bound terminally before clustering begins; there is no domain preference in this first metalation stage. Preferential binding of Zn2+ to β- and α-rhMT at the point where βα-rhMT must form clusters is due to a significant decrease in the affinity of βα-rhMT for further Zn2+. The single-domain Zn5-rhMT, in which there are no exposed cysteine sulfurs, is a key component of the metalation pathway because the lower affinities of the two clustered Zn2+ allows donation to apo-enzymes.