Demethylation of monomethylmercury in freshwater and estuarine sediments and in bacterial cultures was investigated with (CH3HgI)-C-14. Under anaerobiosis, results with inhibitors indicated partial involvement of both sulfate reducers and methanogens, the former dominating estuarine sediments, while both were active in freshwaters. Aerobes were the most significant demethylators in estuarine sediments, but were unimportant in freshwater sediments. Products of anaerobic demethylation were mainly (CO2)-C-14 as well as lesser amounts of (CH4)-C-14. Acetogenic activity resulted in fixation of some (CO2)-C-14 produced from (CH3HgI)-C-14 into acetate. Aerobic demethylation in estuarine sediments produced only (CH4)-C-14, while aerobic demethylation in freshwater sediments produced small amounts of both (CH4)-C-14 and (CO2)-C-14. Two species of Desulfovibrio produced only traces of (CH4)-C-14 from (CH3HgI)-C-14, while a culture of a methylotrophic methanogen formed traces of (CO2)-C-14 and (CH4)-C-14 when grown on trimethylamine in the presence of the (CH3HgI)-C-14. These results indicate that both aerobes and anaerobes demethylate mercury in sediments, but that either group may dominate in a particular sediment type. Aerobic demethylation in the estuarine sediments appeared to proceed by the previously characterized organomercurial-lyase pathway, because methane was the sole product. However, aerobic demethylation in freshwater sediments as well as anaerobic demethylation in all sediments studied produced primarily carbon dioxide. This indicates the presence of an oxidative pathway, possibly one in which methylmercury serves as an analog of one-carbon substrates.