H2 thresholds have been widely used to demonstrate the success of intrinsic bioremediation, however multiple problems exist in obtaining and interpreting H2 field data. Acetate and H2 play similar roles in the metabolism carried out by anaerobic microorganisms, and acetate thresholds have been observed in anaerobic subsurface environments. However, there is little understanding of the factors controlling acetate thresholds. This research used an integrated experimental study of pure cultures and environmental samples, along with microbial respiration modeling, to improve our understanding of acetate thresholds in various terminal electron accepting processes (TEAPs). The results demonstrated that acetate thresholds in pure cultures do not necessarily follow thermodynamic trends, as reported in previous studies, and the model evaluations under PCE-dechlorinating and Fe(III)-reducing conditions revealed that kinetics play a greater role in controlling acetate thresholds in these TEAPs. Acetate thresholds measured in the environmental samples were influenced by the initial acetate concentrations. The results of this study improve our understanding of the factors influencing acetate thresholds in pure and mixed cultures and suggest that acetate thresholds may be a useful component of bioremediation monitoring programs.