The structures of mycotrienins I and II have been determined mainly by their NMR spectral analysis. Mycotrienins are unique ansamycin antibiotics containing a 21-membered macrocyclic lactam ring and a cyclohexylcarbonyl moiety. The labeling experiments with sodium [1-13C]acetate and sodium [1-13C]propionate revealed that six acetate units and two propionate units were incorporated into the molecule of mycotrienin I. In the preceding paper, we reported the production, isolation, physicochemical properties and biological activities of mycotrienins I (MTN-I) and II (MTN-II), together with the taxonomy of the producing organism, Streptomyces rishiriensis T-23. This report presents the structural elucidation and biosynthesis of the antibiotics in detail; a preliminary communication of our work has been presented. Independently, ZEECK and his associates reported the structures of ansatrienins A and B in his recent communication, which have the same structures as mycotrienins I and II, respectively, except for the stereochemistry of alanine.