The biosynthetic origin of antibiotic A10255 was investigated using 14C- and 13C-labeled amino acids. DL-[1-(13)C]Serine labeled 15 of the 17 amino acid residues present in A10255G. These included the oxazole, thiazole, dehydroalanine, masked glycine, masked alanine and pyridine moieties. The same 15 residues labeled by serine were labeled by [2-(13)C]glycine, apparently by conversion of the glycine to [2,3-(13)C]serine. Formation of the pyridine ring occurred via a C3 to C3 condensation of two serines. The results indicated origin of the masked alanine from alanine; the masked glycine from glycine; the thiazole residues from cysteine; and the threonine, masked dehydrobutyrine, masked dehydronorvaline and masked dehydroleucine residues from threonine. L(-)[CH3-(13)C]Methionine labeled the methyl carbon of the masked dehydronorvaline moiety in factor B and the two methyl carbons of the masked dehydroleucine moiety in factor E. The results demonstrate that A10255 originates exclusively from amino acids in a manner similar to the closely related thiopeptide antibiotics nosiheptide and thiostrepton.