Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290±8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90℃, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100℃ was 4h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. Km values of the recombinant enzyme are comparable for both substrates: 0.2mM for L-glutamate and 0.53mM for 2-oxoglutarate. The enzyme was activated by heating at 80℃ for 1h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.