The build-up of methane in the hypolimnion of the eutrophic Lake Rotsee (Lucerne, Switzerland) was monitored over a full year. Sources and sinks of methane in the water column were characterized by measuring concentrations and carbon isotopic composition. In fall, high methane concentrations (up to 1 mM) were measured in the anoxic water layer. In the oxic layer, methane concentrations were much lower and the isotopic composition shifted towards heavy carbon isotopes. Methane oxidation rates peaked at the interface between oxic and anoxic water layers at around 8-10 m depth. The electron balance between the oxidants oxygen, sulphate, and nitrate, and the reductants methane, sulphide and ammonium, matched very well in the chemocline during the stratified season. The profile of carbon isotopic composition of methane showed strong indications for methane oxidation at the chemocline (including the oxycline). Aerobic methane oxidizing bacteria were detected at the interface using fluorescence in situ hybridization. Sequencing the responsible organisms from DGGE bands revealed that aerobic methanotrophs type I closely related to Methylomonas were present. Sulphate consumption occurred at the sediment surface and, only towards the end of the stagnation period, matched with a zone of methane consumption. In any case, the flux of sulphate below the chemocline was not sufficient to oxidize all the methane and other oxidants like nitrate, iron or manganese are necessary for the observed methane oxidation. Although most of the methane was oxidized either aerobically or anaerobically, Lake Rotsee was still a source of methane to the atmosphere with emission rates between 0.2 mg CH(4) m(-2) day(-1) in February and 7 mg CH(4) m(-2) day(-1) in November.