On-line in situ fluorescence measurements of the methanogenic fermentation were conducted with reactors receiving either glucose or a mixture of volatile fatty acids as the substrate. The reactors were perturbed from steady-state conditions in order to assess the response of fluorescencemonitoring probes. Two fluorescence-monitoring probes were evaluated over a period of 8 months; they performed in a consistent manner, and their response was not significantly affected by the changes in pH and redox potential encountered during routine reactor operation. A commercially available probe, designed to measure NAD(P)H, dem- onstrated particular promise for detecting imbalance caused by the entry of air, inhibitor addition and was capable of distinguishing between different substrates. This fluorescencemonitoring probe detected imbalance more rapidly than other on-line measurements such as pH, Eh, or gas production, or off-line measurements such as volatile fatty acid concentration or gas composition. An experimental f luorescence-monitoring probe, designed to measure coenzyme F420, also showed some promise in this regard. The response of the fluorescence-monitoring probes also revealed details of the metabolic routes in the reactors and the probes represent a useful research tool. For example, a failure to observe the characteristic response of the NAD(P)H- monitoring probe to formate addition during the metabolism of acetate, propionate, or glucose strongly suggests that any formate liberated during their catabolism is degraded via a different route to exogenously added formate.