We used stable isotope analysis to investigate dietary differences between 2 sympatric species of tubicolous chironomid larvae, Chironomus plumosus and C. anthracinus, from the profundal sediments of 6 eutrophic lakes in the UK and Germany. We found striking variation between lakes in both δ¹³C (−29.8 to −56.3‰) and δ¹⁵N (−7.8 to 14.7‰) in the 2 chironomid species. Moreover, C. plumosus was consistently depleted in both ¹³C and ¹⁵N relative to C. anthracinus. Our data support previous reports of interspecific dietary variations between the 2 species, which suggest niche separation partly on the basis of diet. However, reported differences in the feeding modes of the 2 species could not explain the extreme ¹³C- and ¹⁵N-depletion observed in chironomid larvae from several of the lakes. We suggest that the low δ¹³C signatures result from the ingestion of methanotrophic bacteria and subsequent incorporation of biogenic methane-derived C. Further, the chironomid larvae enhance methanotrophic activity via bioturbation of the surrounding sediment. Significant isotope differences between the 2 species may result from their variable tube morphologies or physiology.