In this paper, it was first shown that under various conditions of nitrogen supply therifamycin yield was positively correlated with the mycelial specific activity of glutaminesynthetase (GS), then the enhancing effect of glutamine, the product of GS, on rifamycin bio-synthesis was demonstrated with resting cell system. The stimulatory effect of glutaminewas more pronounced than that of glutamate, and not reduced by a GS specific inhibitor, DON.However, the increase in yield brought about by glutamate, and by asparagine was stronglyinhibited by this inhibitor. Glutamine-CO~(15)NH_2 and glutamato-α-~(15)NH_2 were synthesized andcompared for the incorporation of ~(15)N into rifamycin. It was found that the amide nitrogenwas incorporated to a much greater extent than the α-amino nitrogen, showing that glutaminewas a direct precursor of the nitrogen atom in rifamycin. In addition, synthesis of A-32(C_7N), an intermediate secreted by an inactive mutant rif 1, was also greatly stimulatedby glutamine, and the synthetic C_7N was found to be able to stimulate the biosynthesis ofrifamycin. Based upon the above results, the route of incorporation of nitrogen atom into rifamycinis summarized as follows: In this paper, it was first shown that under various conditions of glutamine, the product of GS, on rifamycin bio-synthesis had been correlated with the mycelial specific activity of glutaminesynthetase (GS) with resting cell system. The stimulatory effect of glutamine was more pronounced than that of glutamate, and not reduced by a GS specific inhibitor, DON. However, the increase in yield brought about by glutamate, and by asparagine was strongly inhibited by this inhibitor. Glutamine- It was found that the amide nitrogen was incorporated into a much greater extent than the α-amino nitrogen (CO) (15) NH_2 and glutamato-α- (15) NH_2 were synthesized and compiled for the incorporation of ~ (15) N into rifamycin , showing that glutamine was a direct precursor of the nitrogen atom in rifamycin. In addition, synthesis of A-32 (C_7N), an intermediate secreted by an inactive mutant rif 1, was also greatly stimulatedby glutamine, and the synthetic C_7N was found to be able to stimulate the biosynthesis ofrifamycin. Based upon the above results, the route of incorporation of nitrogen atom into rifamycin is summarized as follows: