Summary. Using Ross's modification of Hutner's method for quantitative determination of vitamin B12 with Euglena gravities var. bacillaris, experiments were carried out which showed that the supernatant from a centrifuged vigorously growing Euglena culture contains a thermolabile factor, which has a growth‐inhibiting effect on Euglena cells when they are transferred to a freshly prepared medium containing vitamin B12. Density and uniformity of growth in the tubes inoculated are increased by washing and diluting the Euglena culture used for inoculation. The sensitivity and the accuracy of the method are thereby increased.