The genetic requirements for the transcription of glnA, encoding the major glutamine synthetase in a rifamycin SV-producing Amycolatopsis mediterranei strain, U32, were investigated. Primer extension experiments showed that the promoter of U32 glnA (pglnA) was likely to have two transcription initiation sites: P1 and P2, located 157 and 45 nucleotides (nt) upstream of the translational start codon, respectively. Gel mobility shift and DNase Ⅰ footprinting analyses revealed a 30 bp cis-element located at 45 to 75 nt downstream of P1, or 38 to 68 nt upstream of P2. The sequence of the cis-element displayed high similarity to the corresponding regions of pglnA from Streptomyces coelicolor and S. roseosporus. With xylE as a reporter gene, the expression levels of U32 pglnA and its deletion derivatives under different nitrogen-source conditions were analyzed by detecting the catechol dioxygenase activities in S. lividans TK54, S. coelicolor J508 and S. coelicolor FS10 (glnR mutant). These in vivo studies showed that the activation of U32 pglnA in S. coelicolor required GlnR, and its binding to the U32 pglnA was further confirmed by the gel mobility shift assay. Cloning and heterologous expression of the U32 glnR allowed us to detect the in vitro interaction between the U32 GlnR and the corresponding pglnA cis-element. Further evidence shown by in vivo glnR inactivation and complementation indicated that GlnR is essential for the active transcription of glnA in U32.