This report describes the isolation of novel A-500359 analogues from the culture broth of Streptomyces griseus SANK 60196 and 13C-incorporation studies of A-500359 A to reveal the biosynthetic pathway of A-500359 derivatives. As a result, A-500359 M-3 and J were isolated as novel analogues. The former, isolated from a culture broth fed with unnatural amino acids, was a novel amino acid adduct of A-500359, and the latter was found to be a putative precursor of all A-500359 derivatives, on the basis of the structure. Moreover, 13C-incorporation studies revealed the origin of every carbon atom of A-500359 A. From these results, it was revealed that the core skeleton of A-500359 was biosynthesized from uridine and phosphoenolpyruvate in the same manner as for polyoxin, a nucleoside antibiotic. Moreover, the uronic acid and aminocaprolactam moiety was derived from hexose and lysine, respectively, and two methyl groups of A-500359 A were derived from methionine.