Phenylalanine tRNA identity has been determined in the bacteria and the eukaryote system, but remains unknown for the archaea system. To investigate the molecular recognition mechanism of phenylalanine tRNA by phenylalanyl-tRNA synthetase from hyperthermophilic and aerobic archaeon, Aeropyrum pernix K1, various mutant transcripts of phenylalanine tRNA prepared by an in vitro transcription system were examined by overexpressed A. pernix phenylalanyl tRNA synthetase. The results indicated that anticodon nucleotides G34, A35 and A36, discriminator base A73 and G20 in the variable pocket were base-specifically recognized by A. pernix phenylalanyl-tRNA synthetase.