In actinomycetes, two main regulators, the OmpR-like GlnR and the TetR-type AmtR, have been identified as the central regulators for nitrogen metabolism. GlnR-mediated regulation was previously identified in different actinomycetes except for members of the genus Corynebacterium, in which AmtR plays a predominant role in nitrogen metabolism. Interestingly, some actinomycetes (e.g. Streptomyces avermitilis) harbour both glnR- and amtR-homologous genes in the chromosome. Thus, it will be interesting to determine how these two different types of regulators function together in nitrogen regulation of these strains. In this study, AmtRsav (sav_6701) in S. avermitilis, the homologue of AmtR from Corynebacterium glutamicum, was functionally characterized. We showed, by real-time reverse transcription (RT)-PCR (qPCR) in combination with electrophoretic mobility shift assays (EMSAs), that gene cluster sav_6697-6700 encoding a putative amidase, a urea carboxylase and two hypothetical proteins, respectively, and sav_6709 encoding a probable amino acid permease are under the direct control of AmtRsav. Using approaches of comparative analysis combined with site-directed DNA mutagenesis, the AmtRsav binding sites in the respective intergenic regions of sav_6700/6701 and sav_6709/6710 were defined. By genome screening coupled with EMSAs, two novel AmtRsav binding sites were identified. Taken together, AmtRsav seems to play a marginal role in regulation of nitrogen metabolism of S. avermitilis.