The ansatrienins produced by Streptomyces collinus ssp. collinus (Lindenbein) strain Td 1982 represent a new type of benzoquinoid ansamycin antibiotic. Ansatrienin B, the hydroquinone of A, seems to be identical with mycotrienin described in 1967, and mycotrienins I and II isolated from Streptomyces rishiriensis have the same structures as ansatrienin A (1) and B except for the configuration of alanine. This communication reports the isolation and structural data of two new ansatrienins, A2 (2) and A3 (3). The ansatrienin complex was extracted from the mycelium of Streptomyces collinus with acetone, oxidized, and separated by PTLC (silica gel, chloroform-methanol 96:4) and HPLC (silica gel column, n-hexane-ether-acetone 5:4:1) to obtain A2 and A3 as yellow amorphous powders (4 mg/liter culture broth each). They are isomers with slightly different physicochemical properties but similar chromophoric systems to ansatrienin A, as indicated by IR and UV spectra. Structural analysis (EI-MS, 1H NMR, hydrolysis, reduction) showed they differ from ansatrienin A in the N-acyl alanine side chain: A2 has a 2-methylbutyryl residue (yielding 2-methylbutyric acid upon hydrolysis) and A3 has an isovaleryl residue (yielding isovaleric acid). Reduction with LiAlH4 afforded ansatrienol A identical to that from ansatrienin A. Antifungal activity tests showed A3 is considerably more active against fungi than A1 and A2. Hydrolysis and esterification experiments revealed L-alanine is an essential part of the ansatrienins for biological activity, and the structure-activity relationship differs from maytansinoids.