The application of a thin agar plate technique to the cup-plate method for the assay of folic acid resulted in a marked increase in the sensitivity, although it incured a lowering of the reproducibility, that is, the precision of the method. Thus, the cup-plate method using a thin ager plate was capable of determining of folic acid in biological materials containing a small quantity of the vitamin and it was effective for improvement of the recovery rates of the added vitamin to these materials. Some of them, however, were unsatisfactory in the recoveries and the dose-response lines for the samples were not parallel from that for standard folic acid solution. In order to eliminate such disadventages encountered in the plate method, the test solutions from natural products, free from the factors affecting the assay results must be obtained.