Demethylation of monomethylmercury in freshwater and estuarine sediments and in bacterial cultures was investigated with ¹⁴CH₃HgI. 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 ¹⁴CO₂ as well as lesser amounts of ¹⁴CH₄. Acetogenic activity resulted in fixation of some ¹⁴CO₂ produced from ¹⁴CH₃HgI into acetate. Aerobic demethylation in estuarine sediments produced only ¹⁴CH₄, while aerobic demethylation in freshwater sediments produced small amounts of both ¹⁴CH₄ and ¹⁴CO₂. Two species of Desulfovibrio produced only traces of ¹⁴CH₄ from ¹⁴CH₃HgI, while a culture of a methylotrophic methanogen formed traces of ¹⁴CO₂ and ¹⁴CH₄ when grown on trimethylamine in the presence of the ¹⁴CH₃HgI. 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.