Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl‐tRNAAsn amidotransferase (Asp‐AdT) capable of forming Asn‐tRNAAsn [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln‐tRNAGln as well as Asn‐tRNAAsn. Thus, the T. thermophilus tRNA‐dependent AdT is a dual‐specific Asp/Glu‐AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy‐terminal amino acids of the GatA subunit only inhibits the Asp‐AdT activity, leaving the Glu‐AdT activity of the mutant AdT unaltered; this shows that Asp‐AdT and Glu‐AdT activities can be mechanistically separated.