Purified yeast thiaminokinase was used for enzymic synthesis of thiamine pyrophosphate (TPP) from adenosine triphosphate (A-PPP)-[gamma]-P32 and thiamine. The product of P32-labeled TPP possessed approximately the same radioactivity ratio of [beta]-P/[alpha]-P (11.5) as that of [gamma] -P/[beta]-P of A-PPP-[gamma]-P32. The following reaction mechanism is offered: Thiamine + A-PPP32 [forward arrow] T-PP32 + A-P.