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Otolith stable carbon isotope ratios provide a unique and widely applicable environmental record. Unfortunately, uncertainty regarding the proportion of otolith carbon that derives from metabolized food versus dissolved inorganic carbon (DIC) in the water currently limits utilization of this marker. We manipulated the δ¹³C of food and ambient DIC in a factorial design with juvenile rainbow trout (Oncorhynchus mykiss). At the activity levels and total metabolic rates characteristic of fish in this study, 17% (±3% standard error, SE) of otolith C was metabolically derived, while >80% was derived from DIC in ambient water. We also estimated isotopic enrichment factors associated with physiological carbon transformations by measuring δ¹³C of blood and endolymph (which closely tracked otolith δ¹³C). There was substantial depletion in ¹³C of blood relative to C sources (ϵ_{blood sources} =  16.9 ± 1.1 SE), but …