Two carbons, carbon-2 and one of carbons-3 to -5, of lysine seemed likely to be incorporated into one of carbon of the chromophore moiety of formycin. 2. From the results of radioisotopic studies with glutamate, gamma-amino-n-butyrate or organic acids related to tricarboxylic acid cycle, the important role of glutamate in the biosynthesis of formycin was strongly suggested. 3. The incorporation of nitrogen(s) of lysine into three nitrogens, including two nitrogens of pyrazole ring, of formycin was suggested by mass spectroscopy. 4. Ribose was estimated as a direct precursor moiety of formycin, whereas the biosynthesis of ribose was shown to occur via the pathway other than hexose monophosphate shunt or glucuronate pathway. 5. In replacement culture, the salvage synthesis of formycin from its chromophore moiety was not observed and it was also evident that the chromophore moiety or pyrazofurin (pyrazomycin) inhibited the biosynthesis of formycin.
