CuA is a dinuclear mixed-valence center located in subunit 2 of the ba(3)-type cytochrome oxidase from Thermus thermophilus. The assembly of this site within the periplasmic membrane is believed to be mediated by the copper chaperones Sco and/or PCuAC, but the biological mechanisms are still poorly understood, thereby stimulating interest in the mechanisms of CuA formation from inorganic ions. The formulation of the CuA center as an electron-delocalized Cu(1.5)-Cu(1.5) system implicates both Cu(II) and Cu(I) states in the metalation process. In earlier work we showed that selenomethionine (SeM) substitution of the coordinated M160 residue provided a ligand-directed probe for studying the copper coordination environment via the Se XAS signal, which was particularly useful for interrogating the Cu(I) states where other spectroscopic probes are absent. In the present study we have investigated the formation of mixed-valence CuA and its M160SeM derivative by stopped-flow UV-vis, EPR, and XAS at both Cu and Se edges, while the formation of fully reduced di-Cu(I) CuA has been studied by XAS alone. Our results establish the presence of previously undetected mononuclear intermediates and show important differences from the metalation reactions of purple CuA azurin. XAS spectroscopy at Cu and Se edges has allowed us to extend mechanistic inferences to formation of the di-Cu(I) state which may be more relevant to biological CuA assembly. In particular, we find that T. thermophilus CuA assembles more rapidly than reported for other CuA systems and that the dominant intermediate along the pathway to mixed-valence is a new green species with λ(max) = 460 nm. This intermediate has been isolated in a homogeneous state and shown to be a mononuclear Cu(II)-(His)(Cys)(2) species with no observable Cu(II)-(Met) interaction. Reduction with dithionite generates its Cu(I) homologue which is again mononuclear but now shows a strong interaction with the Met160 thioether. The results are discussed within the framework of the "coupled distortion" model for Cu(II) thiolates and their relevance to biological metalation reactions of the CuA center.