The activation of brewer's yeast apotransketolase with divalent cations was observed effectively in a low thiamine pyrophosphate concentration. Relative activities were observed in the order of Ca^<2+>>Mn^<2+>>Mg^<2+>>Cd^<2+> and no activity with Zn^<2+>, Cu^<2+>. Holotransketolase which was reconstituted with Ca^<2+> or Mn^<2+> showed negligible resolution in gelfiltration (Sephadex G-25), but that reconstituted with Mg^<2+> or Cd^<2+> lost their original activities in gelfiltration and reactivated by the incubation with thiamine pyrophosphate and Mg^<2+> as high as 38%, 51% respectively. The inhibition of transketolase with Zn^<2+> was removed by means of gelfiltration, while that with Cu^<2+> was not removable.