It was found that an extra exon exists in the first intron of glutamine synthetase gene, generated by means of alternative splicing. Inclusion of this exon decreased the translation of glutamine synthetase (GS) in human, dog, and mouse. When translated in vitro with the canine GS transcript containing the exon, we obtained two different species of GS enzymes. Besides the known 45-kDa protein, the extended form of GS was identified with additional 40 amino acids on its N-terminal end. An upstream ATG in the extra exon served as a translation initiator for the long form of GS. When the long transcript was translated in vivo in animal cells, only the long GS was expressed. On the other hand, the long GS is less predominant relative to the short one in canine tissues including brain and liver. Subcellular fractionation of canine brain revealed that the long GS is present in all cellular compartments as is the short one, which is consistent with fluorescence microscopy data obtained with green fluorescent protein fused to GS. The short (SGS) and long (LGS) forms of canine GS were purified in Escherichia coli and shown to have similar Km values for l-glutamate and hydroxylamine. However, the Km values for ATP were slightly altered, 1.3 and 1.9 mm for the short and long GSs, respectively. The Kis for l-methionine-S-sulfoximine (MSOX), a highly potent ATP-dependent inactivator of GS, were considerably different such that the values are 0.067 and 0.124 mm for the short and long forms, respectively. When the intrinsic fluorescences of tryptophans were monitored upon bindings of chloride and metal ions without any effect on the oligomeric state, the pattern of quenching in LGS was significantly different from that of SGS. Taken together, the N-terminal extension in the long isoform of GS induces a conformational change of core enzyme, leading to a change in affinity to its substrates as well as in the effector-induced conformational alterations.