An assay method for determining of the forms of the thiamine in bakers' yeast was investigated. Inactivation of the phosphatases in yeast by heating before extraction was found to be most suitable. Using this method, the thiamine in yeast was proved to be all in an esterified form. Further, the thiamine formed from the pyrimidine and thiazole moieties by living yeast cells was also estimated. The cells washed with water converted the esterified thiamine to the free form during incubation. This conversion to the free form was less marked in unwashed cells.