Herbimycin is a new potent herbicidal ansamycin antibiotic isolated from the cultured filtrate of Streptomyces hygroscopicus AM-3672. Structure 1 is assigned to herbimycin based on 1H- and 13C-nuclear magnetic resonance (nmr), infrared (ir), ultraviolet (uv) spectra, chemical transformation (reductive acetylation to compound 3), and biosynthetic study using 13C-labeled propionate. The molecular formula C30H42N2O9 for herbimycin (1) was established from elemental analysis and high-resolution mass spectrometry (M+, m/e 574) as reported earlier. The uv spectrum (MeOH) shows characteristic absorption maxima at 270 nm (ε 20090) and 392.5 nm (ε 1650), indicating the presence of a benzoquinone chromophore. 1H- and 13C-nmr spectra indicate that herbimycin is structurally similar to geldanamycin (2), an ansamycin antibiotic. Functional groups including OCONH2, CONH, four OCH3, benzoquinone carbonyls, two K-methyl protons, two methyl protons attached to double bond, a methylene carbon, conjugated diene protons, and an isolated double bond proton were deduced from spectral data. The substitution pattern of the benzoquinone nucleus was deduced from 1H-nmr spectral evidence (two protons at δ 6.60 and 7.30 assigned to α- and α'-positions of the quinone carbonyl) and off-resonance 13C-nmr (quinone carbonyl carbon at δ 183.9 as triplet due to long-range 13C-1H coupling). The structure of the ansachain moiety was confirmed by proton spin decoupling experiment, similarity in 13C chemical shifts with geldanamycin, and biosynthetic study (13C-labeled propionate feeding resulted in strong enrichment in four carbon signals at δ 168.7, 79.2, 130.1, and 34.0 in herbimycin, concordant with the biosynthetic pattern of geldanamycin). Thus, it was concluded that structure 1 is the most suitable for herbimycin.