Mycotrienins are unique benzoquinoid ansamycin antibiotics produced by Streptomyces rishiriensis T-23. In previous studies, we described the isolation, characterization and structural elucidation of mycotrienins I (1) and II (2) and two minor components, mycotrienols I and II. Independently, ZEECK et al. isolated closely related compounds named ansatrienins A and B (differing in the stereochemistry of alanine) and their minor components ansatrienins A2 and A3 (wherein the cyclohexyl group of ansatrienin A is replaced by a 2-methylbutyryl group and an isovaleryl group, respectively). Further screening for minor components of mycotrienins resulted in the isolation of two metabolites named 22-O-methylmycotrienin II (3) and 19-deoxymycotrienin II (4). This article reports their isolation (using the same fermentation procedure as for mycotrienins and purification methods summarized in Fig. 2) and structural elucidation (via 13C NMR, 1H NMR, SIMS, EIMS, nuclear Overhauser effect, and chemical oxidation data). The structure of 3 was elucidated as the 22-O-methyl derivative of 2, and the structure of 4 was determined as the 19-deoxy derivative of 2. Cytotoxicity tests against L-5178Y cells in vitro showed the minimum inhibitory concentrations of 3 and 4 were almost identical (0.11 μg/ml), which were three to four times as active as 2, suggesting that the substituent pattern on the benzene nucleus in mycotrienins is important to their biological activity.