The enzyme encoded by Methanococcus jannaschii open reading frame (ORF) 0490 was purified and characterized. It was shown to be an NADPH-dependent [lactate dehydrogenase (LDH)-like] L-malate dehydrogenase (MalDH) and not an L-lactate dehydrogenase, as had been suggested previously on the basis of amino acid sequence similarity. The results show the importance of biochemical data in the assignment of ORF function in genomic sequences and have implications for the phylogenetic distribution of members of the MalDH/LDH enzyme superfamilies within the prokaryotic kingdom.