Oxidative stress is a state involving an imbalance of reactive oxygen species in a cell and is linked to a variety of diseases. The metal-binding protein metallothionein (MT) may play a role in protection due to its high cysteine content. Many studies have shown that oxidative stress will cause MT to both form disulfide bonds and release bound metals. However, studies on the more biologically relevant partially metalated MTs have been largely neglected. Additionally, most studies to date have used spectroscopic methods that cannot detect specific intermediate species. In this paper, we describe the oxidation and the subsequent metal displacement pathway of fully and partially metalated MTs with hydrogen peroxide. The rates of the reactions were monitored using electrospray ionization mass spectrometry (ESI-MS) techniques, which resolved and characterized the individual intermediate M(SH)MT species. The rate constants were calculated for each species formation. Through ESI-MS and circular dichroism spectroscopy, it was found that the three metals in the β-domain were the first to be released from the fully metalated MTs. The Cd(II) in the partially metalated Cd(II)-bound MTs rearranged to form a protective Cd4MT cluster structure upon exposure to oxidation. The partially metalated Zn(II)-bound MTs oxidized at a faster rate as the Zn(II) did not rearrange in response to oxidation. Additionally, density functional theory calculations showed that the terminally bound cysteines were more negative and thus more susceptible to oxidation than the bridging cysteines. The results of this study highlight the importance of metal-thiolate structures and metal identity in MT's response to oxidation.