Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Clostridium thermoaceticum catalyzes (i) the synthesis of acetyl-CoA from a methylated corrinoid protein, CO, and coenzyme A and (ii) the oxidation of CO to CO2. CO oxidation occurs at a Ni- and FeS-containing center known as cluster C. Electrons are transferred from cluster C to a separate metal center, cluster B, to external acceptors like ferredoxin. In the work described here, we performed reductive titrations of CODH/ACS with CO and sodium dithionite and monitored the reaction by electron paramagnetic resonance (EPR) spectroscopy. We also performed pre-steady-state kinetic studies by rapid freeze-quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics. Redox titrations of CODH/ACS revealed the existence of a UV-visible and EPR-silent electron acceptor denoted center S that does not appear to be associated with any of the other metal centers in the protein. Our results support the previous proposals [Anderson, M. E., & Lindahl, P. A. (1994) Biochemistry 33, 8702-8711; Anderson, M. E., & Lindahl, P. A. (1996) Biochemistry 35, 8371-8380] that the Cred2 form of cluster C is two electrons more reduced than the Cred1 form. The combined results from titrations and pre-steady-state studies were used to formulate a mechanism for CO oxidation, composed of the following steps (i) CO binding to the [Cred1,Box, Xox] state to yield a Cred1-CO complex; (ii) two-electron reduction of Cred1 to Cred2 concerted with CO2 release; (iii) binding of a second CO molecule to the [Cred2,Box,Xox] state to form a Cred2-CO complex; (iv) electron transfer from Cred2-CO to cluster B to form [Cred2,Bred,Xred] with concerted release of the second CO2. Step iii competes with internal electron transfer from Cred2 to Box and Xox. At high CO concentrations, step iii is favored, whereas at low concentrations, only one CO molecule per turnover binds and undergoes oxidation. Closure of the catalytic cycle involves electron transfer from reduced enzyme to an electron acceptor protein, like ferredoxin. Xox is a yet-uncharacterized electron acceptor that may be an intermediate in the reduction of center S. The Cred2 state appears to be the predominant state of cluster C during steady-state turnover. The rate-determining step for the first half-reaction is step iv, while during steady-state turnover, it appears to be electron transfer to external electron acceptors.