The structure of a new metabolite T-2 toxin (iso-TC-1) has been established as 3,15-diacetoxy-4-hydroxy-8(3-methyl-3'-hydroxy-butyryloxy)-lZ,l3-epoxytrichothec-9-ene. The compound is an isomer of TC-1 (a recently isolated T-2 derivative) in which the hydroxy and acetoxy groups at the C-3 and C-4 positions, respectively, are reversed. Direct probe analysis by electron impact (EI) of the underivatized iso-TC-1, as well as EI, positive chemical ionization (CI) in methane, and positive CI in ammonia of its trimethylsilylether or trifluoroacetate provided evidence to support the structure assignment of the new metabolite. The mass spectra of iso-TC-1 were compared with those of TC-1, T-2 toxin and iso-T-2 toxin (the isomer of T-2 toxin having reversed substituents at C-3 and C-4) with regard to molecular weight and fragments involving the substituents at C-3, C-4, C-8 and C-15. Although the two isomers, TC-1 and iso-TC-1, were not easily resolved by thin layer chromatography (TLC), a very good separation of their trimethylsilyl and trifluoroacetate derivatives was obtained by capillary gas chromatography. Acetylation of TC-1 or iso-TC-1 gave the same product. Iso-TC-1 is one of the main products of T-2 metabolism in the cow (more abundant than TC-1) and is found in the urine.