Stable carbon isotope analysis of chironomid larvae gave rise to the hypothesis that methane-oxidizing bacteria can provide an important food source for higher trophic levels in lakes. To investigate the importance of the methane cycle for the larval stable carbon signatures, isotope analysis and microbiological and biogeochemical investigations were combined. The study was based on comparison of a dimictic lake (Holzsee) and a polymictic, shallow lake (Großer Binnensee), both located in northern Germany. Both lakes are inhabited by Chironomus plumosus larvae, which exhibited a stronger ¹³C-depletion in Holzsee than in Großer Binnensee, indicating a greater contribution of methane–carbon in the former. Indeed, the processes involved in the microbial methane cycle were found to be more active in Holzsee, showing higher potential methane production and methane oxidation rates. Consistently, cell numbers of methane-oxidizing bacteria were with 0.5 − 1.7× 10⁶ cells about one order of magnitude higher in Holzsee than in Großer Binnensee. Molecular analysis of the microbial community structure revealed no differences in the methanotrophic community between the two lakes, with a clear dominance of type I methanotrophs. The methanogenic population seemed to be adapted to the prevailing substrate in the respective lake (H₂/CO₂ in Holzsee and acetate in Großer Binnensee), even though differences were minor.