Even though lakes only occupy a small percentage of Earth's surface, they represent a natural source of methane, contributing substantially to methane emissions and ultimately to global warming. There is extensive evidence that methane emissions from lakes are mitigated by its oxidation. However, many uncertainties concerning biotic controls on methane release from lakes remain, especially in zones where oxygen is depleted and other electron acceptors might be important. In this study, biological methane oxidation was investigated in the water column of Lake La Cruz, a small karstic lake located in Central Eastern Spain near the city of Cuenca. Permanent stratification and unusually high concentrations of dissolved iron(II) make this lake ideal for studying methane oxidation in the absence of oxygen, particularly the possibility of this process coupled to iron reduction. The physical and chemical properties of Lake La Cruz were investigated, including the presence of methane and possible electron acceptors. Incubation experiments with 13C-methane were carried out to measure methane oxidation rates, test possible electron acceptors and other coupled processes. Highest methane oxidation rates were measured at the oxic-anoxic boundary, where aerobic oxidation dominated. Moreover, the aerobic pathway also seemed relevant below the oxycline, likely triggered by in-situ production of oxygen via photosynthesis. Evidence suggests that nitrate, nitrite and iron also function as electron acceptors for methane oxidation, whereas manganese and sulfate are unlikely oxidants. Methane oxidation coupled to denitrification seemed to proceed in close vicinity to aerobic methane oxidation and was likely limited by nitrate/nitrite availability and competition with denitrifiers. Iron mediated methane oxidation only appeared relevant in lower parts of the lake where both oxygen and nitrate were depleted. Though known aerobic methanotrophs were present and appeared to mediate methane oxidation to some degree, the involvement of unknown groups of aerobic and also anaerobic methane-oxidizers is probable. Methane emissions to the atmosphere are effectively mitigated by a seemingly complex interplay of aerobic and anaerobic processes in Lake La Cruz.