The 1410 bp DNA region (glnA) encoding glutamine synthetase I (GSI) from Streptomyces viridochromogenes was amplified by PCR, cloned and sequenced. The molecular mass of the deduced GSI protein (469 residues) was determined to be 50 kDa. The DNA region showed 90% nucleotide identity with the Streptomyces coelicolor A3(2) glnA gene, but no significant nucleotide sequence similarity with the glnII (GSII) gene of S. viridochromogenes. The chromosomal glnA and glnII genes of S. viridochromogenes were disrupted by site-specific mutagenesis. Neither glnA nor glnII single mutants required glutamine for growth and both were normal in their sporulation. Measurement of the GS activity in cultures grown with different nitrogen sources revealed that GSI (heat-stable) and GSII (heat-labile) were always expressed together, with GSI as the predominant activity. It could be proposed that GSI, but not GSII is inactivated by adenylylation under conditions of nitrogen excess. GSI and GSII activities are inhibited by amino acids and by nucleotides.