Larvae of the chironomid Stictochironomus pictulus were collected from Lake Biwa, central Japan. Both the fatty acid composition of the total lipid fraction and the carbon stable isotope ratios of whole larvae were determined. Larvae showed delta(13)C values of -57.4 per thousand to -62.4 per thousand, similar to the values of methane recorded from the lake sediments. A high level of monounsaturated fatty acids (MUFAs; approximately 50% of total fatty acids) and an extremely low level of n-3 series polyunsaturated fatty acids (PUFAs) in the total lipids of S. pictulus indicated a predominantly bacterial nutrition for this species. Moreover, chironomid tissues contained large amounts of the Type I methanotroph group-specific fatty acid, 16:1(n-8) (approximately 8% of total fatty acids). This is the first time such a fatty acid biomarker has been described from freshwater invertebrates. The data suggest that S. pictulus larvae directly feed upon methanotrophic bacteria.