To clarify the effect of ethanol on thiamine metabolism and activity of transketolase, a thiamine dependent enzyme, in vivo and in vitro experiments were carried out. For the in vivo experiment, rats were separated into four groups: Group 1, thiamine sufficient diet with no ethanol administered; group 2, thiamine sufficient diet and ethanol administered by stomach tube; group 3, thiamine deficient diet with no ethanol administered; group 4, thiamine deficient diet and ethanol administered. After three weeks, the rats were killed, then thiamine levels and the activity of transketolase were assayed. Thiamine concentration in brains and livers of ethanol fed rats were lower than that in control rats. Urinary thiamine was higher in ethanol administered rats than in controls. The increase in the liver transketolase activity by the in vitro addition of cocarboxylase (TPP effect) was higher in group 3 than in group 4 although original transketolase activity was lower in group 4. For the in vitro experiment, various amounts of ethanol or acetaldehyde were added to the supernatant of brain and liver of intact rats, and transketolase activity was determined. Inhibition by acetaldehyde to transketolas was about 10 times higher than that by ethanol.