Glutamate dehydrogenase (GDH; EC 1.4.1.2) catalyzes the reductive amination of 2-oxoglutarate with ammonium and the oxidative deamination of glutamate. In some plants species, GDH is a hexamer and can be separated into seven isoenzymes that are composed of two distinct subunits: a and b. The large number of isoenzymes is the reason why GDH functions are still being intensively researched and widely studied. Until recently, the b subunit of GDH in triticale, a common Polish cereal, was thought to be encoded by the TsGDH1 gene, which can undergo posttranslational modifications and form a heterohexameric enzyme. Here, we report the cloning and molecular characterization of a second glutamate dehydrogenase geneTsGDH2 (encoding a subunits). The TsGDH2 cDNA contains a 1236-bp open reading frame encoding a 411-amino-acid polypeptide with a calculated molecular mass of 44.5 kDa. To clarify the role of TsGDH2 in triticale, we used triticale GDH a-subunit cDNA to generate transgenic A. thaliana lines with increased and decreased GDH activity via alternation of a-subunit levels.