The glutamate dehydrogenase (GDH) gene of a hyperthermophilic archaeon, Thermococcus profundus DT5432, was cloned in Escherichia coli and sequenced. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp encoding a polypeptide of 419 amino acids with a molecular weight of 46,699, The structural gene is preceded by an archaeal promoter consensus sequence with a spacing of 50 bases and is followed by a pyrimidine rich sequence which may function as a transcriptional terminator. The GDH gene was expressed in E. coli under control of the iac promoter. The purified recombinant GDH exhibited pH optima and thermostability identical to those of the intrinsic enzyme; however, it showed slightly higher K-m values for NADP, NADP(+), 2-oxoglutarate and ammonia than the intrinsic enzyme. The NH2-terminal methionine was not removed in E. coli. The derived amino acid sequence is 53% identical to that of GDH from a mesophile, Clostridium difficile. Amino acid sequence alignment between the thermococcal and the clostridial GDHs revealed the presence of significantly more alanine residues and significantly fewer glycine residues in alpha-helical regions, and more proline residues in loop regions, in the former than in the latter. These amino acid differences are located exclusively in the NH2-terminal half (residues 54-253) of the enzyme.