A gene encoding an l-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable l-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 °C. The optimum temperature for l-aspartate dehydrogenation was about 80 °C. The enzyme specifically utilized l-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The K m values for l-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The K m values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the K m values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro- R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal l-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species.