In cyanobacteria, the glutamine synthetase-L-glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) pathway is the major ammonia-assimilating route. The GS of Anabaena doliolum was synthesized more under N2-fixing conditions, followed by ammonium, nitrate, and nitrite as nitrogen sources. The activities of both the glutamine synthetase, Mg2+-dependent biosynthetic and Mn2+-dependent gamma-glutamyl transferase were optimum at pH 7. The active site of the enzyme bears sulfhydryl (-SH) groups, this was confirmed with the -SH group inhibitors, para-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM). The biosynthetic and gamma-glutamyl transferase activities showed specificity for the divalent cations, Mg2+ and Mn2+, respectively. The other divalent cations Co2+, Cu2+, and Ni2+ were poor substitutes. This enzyme also required these divalent cations to stabilize its structure and function under extreme conditions such as high and low temperatures and urea denaturation. The glutamate analog L-methionine-D,L-sulfoximine, inactivated the enzyme, whereas the GOGAT inhibitor, azaserine, had no effect on the enzyme activity. The GS enzyme required de novo protein synthesis.