The argR gene of Streptomyces clavuligerus has been located in the upstream region of argG. It encodes a protein of 160 amino acids with a deduced M(r) of 17117 for the monomer. Transformants containing the amplified argR gene showed lower activity (50%) of the biosynthetic ornithine carbamoyltransferase (OTC) activity and higher levels (380%) of the catabolic ornithine aminotransferase (OAT) activity than control strains. Amplification of an arginine (ARG) box-containing sequence results in a 2- to 2.5-fold derepression of ornithine acetyltransferase and OTC, suggesting that the repressor is titrated out. Footprinting experiments using the pure homologous arginine repressor (AhrC) of B. subtilis showed a protected 38 nt region (ARG box) in the coding strand upstream of argC. The protected region contained two tandemly repeated imperfect palindromic 18-nt ARG boxes. The repressor-operator interaction was confirmed by bandshift experiments of the DNA fragment containing the protected region. By computer analysis of the Streptomyces sequences available in the databases, a consensus ARG box has been deduced for the genus Streptomyces. This is the first example of a clear regulation of an amino acid biosynthetic pathway in Streptomyces species, challenging the belief that actinomycetes do not have a well-developed regulatory system of these pathways.