We report here the cloning and sequencing of Pseudonocardia thermophila JCM3095 genomic DNA clones that encode a nitrile hydratase (NHase). The NHase exhibits high optimum temperature and heat stabilities, and requires Co2+ for high activity. The deduced amino acid sequence of the enzyme consists of a (203 aa, 612 bp) and beta (233 aa, 699 bp) subunits. P. thermophila is a moderate thermophile, and this is the first report of an NHase gene from a thermophile.