Inactivation and initial interrogation of key oxygenase CmmOIV of the biosynthetic pathway of chromomycin A(3) in Streptomyces griseus ssp. griseus revealed that a completely methylated and acetylated prechromomycin is the preferred substrate of this enzyme. This suggests that the three sugar decoration reactions, two O-acetylations and an O-methylation, which were previously believed to occur as the final steps of chromomycin A(3) biosynthesis, indeed take place prior to the CmmOIV reaction. Upon inactivation of CmmOIV, four new compounds accumulated; the fully decorated prechromomycin and its incompletely acetylated precursor along with a diketoprechromomycin-type compound were fully characterized and assayed with CmmOIV.