Rainbow trout Oncorhynchus mykiss in Cayuga Lake, New York, appear to be suffering from a thiamine deficiency because their progeny develop general weakness, loss of equilibrium, and increased mortality, which are prevented by treatment with thiamine. Thiamine status and its effect on adults are unknown. In 2000 and 2002, we captured, tagged, and released 64 and 189 prespawning rainbow trout, respectively, in Cayuga Inlet at a collection weir to evaluate their thiamine status and the effect of thiamine injection (150 nmol/g) on instream migration. Half of the rainbow trout in each year (32 in 2000 and 95 in 2002) were injected with thiamine and half were uninjected; all rainbow trout were released above the weir to continue their upstream migration. By means of electrofishing in 2000, we recaptured significantly more thiamine-injected (N = 7) than uninjected (N = 0) rainbow trout approximately 7.0–9.3 river kilometers upstream from the weir. In 2002, the concentration of thiamine in the muscle of rainbow trout collected above a 1.8-m cascade was significantly higher (mean ± SD = 5.47 ± 5.04 nmol/g; range = 1.0– 13.8 nmol/g; N = 8) than that of rainbow trout collected either above a 1.0-m cascade (1.36 ± 0.71 nmol/g; range = 0.6–3.3 nmol/g; N = 16) or below the cascades (1.20 ± 0.46 nmol/g; range = 0.7–1.9 nmol/g; N = 5). The lowest concentration of thiamine observed in the muscle of rainbow trout collected upstream of the 1.8-m cascade was 1.0 nmol/g, suggesting that the threshold concentration required for rainbow trout to ascend the cascade was no more than that. Analyses of thiamine in the muscle of 26 untagged rainbow trout captured in Cayuga Inlet in 2002 showed that 16 fish (62%) had at least 1.0 nmol/g, which was apparently sufficient to support vigorous migration.