Balhimycin is a recently introduced glycopeptide antibiotic isolated from Amycolatopsis sp. Y-86, 21022 (DSM59081), characterized by a 4-dehydrovancosamine sugar unit (mainly present as ketone hydrate) and exhibiting in vitro and in vivo antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains and many anaerobic organisms. To investigate the biochemical synthesis performance of Amycolatopsis sp. Y-86, 21022 and discover new glycopeptide components, we examined nutrient broths of balhimycin producers. Cultures of Amycolatopsis sp. Y-86, 21022 (as described by S. R. Nadkarni et al.) served as the starting material. The antibacterial compounds were desalted and concentrated using the Diaion HP-20 adsorption resin technique, separated on an SP Spherodex M cation exchanger (Sepracor/IBF, France), and finally purified via preparative HPLC in a 0.05% trifluoroacetic acid/acetonitrile system and crystallization from water/ethanol (Scheme 1). The most important data on the isolated compounds are summarized in Table 1: Glycopeptide 1 was identified as desvancosamine vancomycin using hydrolysis products and degradation of commercial vancomycin; 2 was ureidobalhimycin (1-5% of balhimycin, semisynthesizable from balhimycin and KNCO, with a characteristic KNCO reaction for 4-oxovancosamine); 3 was M43C (glucose as the only sugar component, no KNCO reaction, confirmed by GC-MS, MS, HR-MS, and ¹H NMR); 4 was trace deglucobalhimycin; 6 was rhamnosyl balhimycin (confirmed by hydrolysis products and FAB-MS fragmentation); 7 was N,N-dimethylleucyl balhimycin; 8 was norbalhimycin; 9 was dechloro-balhimycin V (from 10's dehalogenation); and 10 contained two 4-oxovancosamine units. Compound 10's hydrolysis yielded 4-oxovancosamine, which was isolated for NMR analysis via ion-exchange chromatography. MIC data (Table 3) showed the antibacterial activity of these glycopeptides against Staphylococcus aureus strains.