Cytosinine-the nucleoside portion of blasticidin S-and pyridoxal phosphate were incubated with cell-free extracts of Streptomyces griseochromogenes prepared in D₂O. ²H NMR analysis of recovered cytosinine showed it to contain deuterium enrichments at H-4' and H-2'. No exchange was observed with either boiled extract or from cytosinine and pyridoxal phosphate alone. These results reveal the presence of a tautomerase activity that contributes to the net transamination at C-4' in the conversion of cytosylglucuronic acid to blasticidin S, and its discovery supports the role of cytosinine as a biosynthetic intermediate.