Streptomyces hygroscopicus var. jinggangensis produces validamycin, an important antibiotic used in agriculture. It was found that there was a positive correlation between the specific activity of mycelial glutamine synthetase (GS) and validamycin biosynthesis in our laboratory. So, in this paper, the purification, characteristics and regulatory properties of GS are reported. The native enzyme had a molecular weight of approximately 530,000 and was composed of 12 identical subunits, each 43,000. Electronic microscopic examination of preparations negatively stained disclosed that the subunits were arranged in two hexagonal rings that lay one on top of the other in a face-to-face fashion. Mg2+ or Mn2+ was absolutely needed as cofactor for GS activity. The enzyme activity was regulated by feedback inhibition and cumulative feedback inhibition. In addition, it was also regulated through a covalent modification, adenylylation and deadenylylation, suggesting that the covalent modification of GS exists not only in Gram-negative bacteria but also in some Gram-positive bacteria.