McClelland and Montoya (2002) pointed out that the significant 15N-enrichment in glutamate (Glu) along the food web provides a scope for defining trophic position, whereas little isotopic changes in phenylalanine (Phe) provide information of nitrogen sources at the base of the food web. In this study, to establish as a tool for precisely estimating the trophic level of organisms, we have investigated the nitrogen isotopic compositions of these amino acids from various cultured and natural organisms including algae, cyanobacteria, zooplankton, rotifer, crab, and fish whose trophic levels are known. Based on these results, we have established an equation obtaining precise trophic level of organisms, employing the following equation: TL=(d15N(Glu)-d15N(Phe)-3.4)/7.6+1 (Chikaraishi et al., submitted). The resulting error is much smaller than that in traditional estimates based on bulk isotope analysis. We have applied the method to several types of samples including formalin-fixed fish samples collected from Lake Biwa where significant eutrophication occurred during the 1960s to 70s. We did not find any significant changes in trophic level of the fish during the eutrophication period. It suggests that the trophic position did not change along the eutrophication, although species composing the ecosystem drastically changed.