Studies were made on pyruvate-oxidase activity in thiamine deficient rats. The enzymatic activity was estimated by measuring the volume of O_2 taken up into a washed liver homogenate. Effects of addition of thiamine diphosphate, DPN, CoA, lipoic acid, ATP, nicotinamide, cytochrome c, glutathione and vitamin B_<12> in various combinations were also examined. Qo_2 (N) value of the enzymatic system was remarkably lower in the deficient rats than in the control. The oxidase in the deficient rats was not activated by the cofactors described above. These results suggest that the low activity of pyruvate-oxidase in thiamine deficient rats is not due to the deficiency of the vitamin as an coenzyme, but rather to insufficient synthesis of the apoenzyme.