An extreme thermophilic archaeon, Aeropyrum pernix K1 possesses two possible threonyl-tRNA synthetase genes. Sequence homology analysis of these genes with other species threonyl-tRNA synthetase showed that the shorter gene did not possess motif-2 and motif-3 of catalytic core that were conserved in class II aminoacyl-tRNA synthetases. On the other hand, the longer gene had almost all amino acids that were expected to be involved in substrate binding and catalytic activity. As a striking feature, it was found that the sequence of the longer threonyl-tRNA synthetase was unique in its quite compact N-terminal domain. This peculiar structure of A. pernix threonyl-tRNA synthetase may suggest one of the hints that can decipher not only the evolutionary position of this archaeon but also the evolutionary process for threonyl-tRNA synthetase. Cross-species aminoacylation experiments showed that threonyl-tRNA synthetase from A. pernix threonylated not only Escherichia coli threonine tRNA having A73 as a discriminator base, but also an extreme halophilic archaeon Haloferax volcanii threonine tRNA possessing U73. These results indicate that A. pernix threonyl-tRNA synthetase does not recognize the discriminator base like E. coli system.