A hyperthermophilic hydantoinase from Methanococcus jannaschii with an optimum growth at 85degreesC was cloned and expressed in E. coli. The recombinant,hydantoinase was petrified by affinity and anion-exchange chromatography and determined to be homotetrameric protein by gel filtration chromatography. The best substrate for the hydantoinase was D,L-5-hydroxyhydantoin, which has the specific activity of 183.4 U/mg. The optimum pH and temperature for the hydantoinase activity was 8.0 and 80degreesC, respectively. The half-life of the hydantoinase was measured to be 100 min at 90degreesC in the buffer containing 500 mM KCl. Manganese ions were the most effective for the hydantoinase activity. Stereospecificity was determined to be L-specific for the 5-hydroxymethylhydantoin and 5-methylhydantoin by chiral TLC. The activity yields as well as the operational stabilities of the thermostable M. jannaschii hydantoinase could be significantly improved by immobilization method.