1. Using M. glutamicus the quantitative analyses were made both on the amino acids excreted into the media and incorporated into the cells at the different levels of biotin.2. The dual role of biotin in relation to the nitrogen metabolism in the glutamic acid fermentation is discussed. The biotin deficiency brought about the increase in the formation of glutamate and the decrease in the utilization of glutamate. 3. Not only the excretion of glutamate but also that of the other amino acids increased under the deficiency of biotin.4. The biotin deficient cells contained less intracellular free amino acids than the biotin rich cells.5. The compositions of intracellular bound amino acids of the biotin rich cells and the biotin deficient cells showed a close resemblance except some amino acids.6. The contents of some of the intracellular bound amino acids changed appreciably with the biotin levels in the growth media. The contents of proline, methionine, tyrosine, lysine, and histidine decreased on lowering the biotin level in the growth medium. This may provide a possible explanation both for the growth regulation and the glutamate accumulation by M. glutamicus under the deficiency of biotin.