Anaerobic oxidation of methane (AOM) is one of the major processes limiting the release of the greenhouse gas methane from natural environments. Here we show that significant AOM exists in the methane zone of lake sediments in natural conditions and even after long-term (ca. 18 months) anaerobic slurry incubations with two stages. Methanogens were most likely responsible for oxidizing the methane, and humic substances and iron oxides are likely electron acceptors to support this oxidation.