Carbon monoxide was a normal trace component of the gases produced during anaerobic sludge digestion. The CO concentration increased in response to perturbing the digestion process by increasing organic loading or adding acetate. Reducing the headspace methane level resulted in higher measured CO concentrations. Accordingly, a thermodynamic relationship was developed by dividing the acetoclastic methane reaction into two half-cell reactions, representing production of and subsequent oxidation of CO. A constant fraction of the total free energy available for acetate conversion to methane was assigned to each half-cell based on the basis of experimental observations. It was determined that approximately 54% of the energy available for acetate conversion to methane was consistently associated with the anaerobic oxidation of CO to carbon dioxide. Estimated values compared well for measured concentrations for both mesophilic and thermophilic digesters operating under steady-state conditions. The thermodynamic relationship, based upon gaseous components only, was manipulated to accurately predict acetate concentration in a digester system subjected to an organic overload that was monitored with an on-line data acquisition system. © 1990, American Chemical Society. All rights reserved.