The purification and some properties of glutamine synthetase (GS) from the mycelium of the basidiomycete Pleurotus ostreatus are described. The enzyme was purified to apparent homogeneity with ion exchange chromatography and a Dyematrex Green A column as the major purification steps. The GS has a molecular weight of 470 kDa and is composed of eight subunits with a molecular weight of 58 kDa. A tetrameric form of the enzyme may also be active. The apparent K-m values for the biosynthetic reaction varied in different mycelial extracts from 2.5 to 3.5 mM and from 0.02 to 0.06 for glutamate and ammonium respectively. In the transferase reaction, K-m values of 48 mM and 6.2 mM were found for L-glutamine and hydroxylamine, respectively. From the divalent cations tested, Mn2+ showed the strongest stimulatory effect both on the transferase and the biosynthetic reaction. ADP was the only nucleotide having an activating effect on the transferase reaction. The biosynthetic reaction was strongly inhibited by AMP and the transferase reaction by carbamoylphosphate. L-Alanine and glycine inhibited both reactions.