Abstract Cell-free protein synthesis, driven by a crude S30 extract from Escherichia coli, has been applied to the preparation of proteins containing unnatural amino acids at specific positions. We have developed methods for inactivating tRNAAsp and tRNAPhe within a crude E. coli tRNA by an antisense treatment and for digesting most of the tRNA within the S30 extract without essentail damage to the ribosomal activity. In the present study, we applied these methods to the substitution of Asp and Phe residues of the HIV-1 protease with unnatural amino acids. With 10 mM Mg2+, the translation efficiency was higher than that with the other tested concentration, and the misreading efficiency was low. The protease mRNA was translated in the presence of an antisense DNA-treated tRNA mixture and 2-naphthylalanyl- and/or p-phenylazophenylalanyl-tRNA. The results suggest that a good portion of the translation products are substituted at all of the seven positions originally occupied by Asp or Phe.