Abstract Bacillus subtilis glutamine synthetase requires high ionic strength for optimal γ-glutamyltransferase activity; 0.4 m K2SO4 added directly to the assay enhances catalytic activity more than 12-fold. Specific monovalent cations modify this enhanced activity, with potassium salts favoring activation more than sodium salts, and rubidium salts intermediate between the two. Lithium antagonizes the stimulatory effects of high ionic strength. In contrast, the biosynthetic activity of glutamine synthetase is inhibited by salts added directly to the assay, the relative inhibition being greatest with potassium salts, least with lithium, and intermediate with sodium. Incubation of glutamine synthetase in buffer with high salt concentrations prior to assay stimulates the transferase activity also, but has no effect on the biosynthetic activity. This latter stimulation of activity is independent of the monovalent cation in the preliminary incubation, but is blocked by Mn2+, and to a lesser extent by Mg2+, when these divalent cations are included in the preliminary incubation buffer. When the enzyme is studied under optimal conditions of preliminary incubation and assay, the Mn2+ and ADP substrate saturation curves are sigmoid, but those of arsenate, glutamine, and hydroxylamine are hyperbolic. The transferase activity is subject to feedback inhibition by tryptophan, AMP, glycine, and alanine. The enzyme in the high salt buffer has altered sulfhydryl reactivity and increased fluorescence with the hydrophobic probe, N-phenyl-N-methyl-2-aminonaphthalene-6-sulfonate, in comparison to enzyme in the low salt buffer. No differences, however, between the high and low salt preparations could be detected in sedimentation velocity and light-scattering experiments. After 17 hours, some dissociation of the enzyme centrifuged in the high salt buffer is demonstrated in sedimentation equilibrium studies; this dissociation was not seen in comparable studies without added salt. Assay in high salt concentrations raises the apparent affinity of the enzyme for the substrates of the transferase assay; such changes may explain partially the marked increase in the transferase activity associated with the high salt environment.