Intermediates in the biosynthesis of blasticidin S and its nucleoside co-metabolites were detected by altering fermentation conditions. Inhibitors of specific types of biochemical reactions that were expected to be involved in blasticidin biosynthesis were fed toStreptomycesgriseochromogenes, in some cases with the inclusion of large quantities of the primary precursors of blasticidin S. The types of reactions and inhibitors used were (1) transaminase (aminooxyacetic acid and 2-methylglutamate), (2) amidotransferase (azaserine and 6-diazo-5-oxo-L-norleucine), (3) arginine biosynthesis (arginine hydroxamate), and (4) methyltransferase (ethionine). These manipulations apparently distorted the pools of precursors and (or) intermediates, and led to substantial accumulations of three known, previously minor, metabolites ofS.griseochromogenes, cytosylglucuronic acid, pentopyranine C, and demethylblasticidin S, and of two new ones, pentopyranone and isoblasticidin S. New cytosyl metabolites were detected by HPLC with photodiode array detection. Fermentations to which arginine hydroxamate and cytosine had been added also produced three aberrant metabolites that were derived from pentopyranone and arginine hydroxamate.