We report the isolation and characterization of a Neurospora crassa glutamine synthetase (GS) mutant altered in one of the two polypeptides (GS alpha) of this enzyme. We used the gln-1bR8 mutant strain that synthesizes only the GS alpha monomer and lacks the GS beta monomer and selected for growth in minimal medium in the presence of alpha-methyl-DL-methionine-SR-sulfoximine (alpha-me-MSO), an inhibitor of GS activity. The GS activity of the gln-1bR8;alpha-me-MSOR strain drastically reduced its transferase activity and only slightly reduced its synthetase activity, and it was resistant to inhibition by alpha-me-MSO and L-methionine-DL-sulfoximine. The mutation that overcame the inhibitory effect of alpha-me-MSO also altered the antigenic, kinetic, and physical properties of GS alpha. The low GS activity of the alpha-me-MSO-resistant strain was compensated for by a higher glutamate/glutamine ratio and a lower glutamate synthase activity, allowing this strain to grow as well as the parental strain. The mutation that conferred resistance to alpha-me-MSO was not linked to the gln-1bR8 mutation, providing direct evidence of the existence of two genes involved with the structure of the two polypeptides of N. crassa GS.