To isolate Streptomyces tendae mutants blocked in the biosynthesis of the nikkomycin nucleoside base 4-formyl-4-imidazoline-2-one, an assay was developed to detect the formation of nikkomycins containing this base during growth on solid medium. The assay is based on the reaction of the 4-formylimidazolone structure of nikkomycins with the aldehyde reagent barbituric acid leading to red-colored products. Among 18,000 N-methyl-N'-nitro-N-nitrosoguanidine treated clones tested in the barbituric acid assay, we isolated one mutant which was incapable of forming any nikkomycins containing the 4-formylimidazolone base (nikkomycins Cx, X and I) but instead produced nikkomycins containing uracil (nikkomycins C, Z and J). In addition, we isolated strains with mutations affecting the biosynthesis of 2-amino-4-hydroxy-4-(5-hydroxy-2-pyridyl)-3-methylbutyric acid, the unusual amino acid of nikkomycins Z, X, J and I. By analyzing colonies derived from single spores or protoplasts of S. tendae Tü901/395, a mutant producing besides nikkomycins Z, X, J and I, also nikkomycins Kz/Kx and Oz/Ox, we obtained strains which only formed nikkomycins Kz/Kx and Oz/Ox with 2-amino-4-hydroxy-4-(2-pyridyl)butyric acid and 2-amino-4-hydroxy-4-(5-hydroxy-2-pyridyl)-butyric acid as amino acids. Mutation of such a strain (Tü901/395-11) by UV365nm in the presence of 8-methoxypsoralen and selection of S-2-aminoethyl-L-cysteine-resistant clones led to the isolation of Tü901/AEC1 and AEC2 which produced exclusively nikkomycins Kz and Kx. According to their nikkomycin spectrum, these strains were blocked at the hydroxylation step occurring at the pyridyl residue during biosynthesis of the nikkomycin amino acid.