The15N/14N ratio of the fish-native organic matter preserved in fish otoliths (or δ15Noto) may allow for reconstruction of fish trophic history and changes in food webs. To support this application, ground-truthing data are needed on the relationships among the δ15N of diet, of fish tissue (e.g., white muscle tissue, δ15Nwmt), and δ15Noto. Using a highly sensitive method for N isotope analysis, δ15Notowas compared with δ15Nwmtin 24 teleost species. Within a species, the difference between δ15Notoand δ15Nwmt(Δδ15No-w) varied little across individuals, confirming the utility of δ15Nototo reconstruct δ15Nwmtchanges for a given species. Across species, δ15Notoand δ15Nwmtwere highly correlated. However, Δδ15No-wvaried systematically across species. Phylogeny, the concentrations of total N and amino acids, and life history were ruled out as the main cause for the observed variation in Δδ15No-w. δ15Notowas lowest relative to δ15Nwmtin species producing larger otoliths. We propose that δ15Notois elevated by isotopically fractionating metabolism of the organic matrix, which is less important when otolith growth is fast and thus when the otolith is large.