We have constructed an opal suppressor system in Escherichia coli to complement an existing amber suppressor system to study the structural basis of tRNA acceptor identity, particularly the role of middle anticodon nucleotide at position 35. The opal suppressor tRNA contains a UCA anticodon and the mRNA of the suppressed protein (which is easily purified and sequenced) contains a UGA nonsense triplet. Opal suppressor tRNAs of two tRNA(Arg) isoacceptor sequences each gave arginine in the suppressed protein, while the corresponding amber suppressors with U35 in their CUA anticodons each gave arginine plus a second amino acid in the suppressed protein. Since C35 but not U35 is present in the anticodon of wild-type tRNA(Arg) molecules, while the first anticodon position contains either C34 or U34, these results establish that C35 contributes to tRNA(Arg) acceptor identity. Initial characterizations of opal suppressor tRNA(Arg) mutants by suppression efficiency measurements suggest that the fourth nucleotide from the 3' end of tRNA(Arg) (A73 or G73 in different isoacceptors) also contributes to tRNA(Arg) acceptor identity. Wild-type and mutant versions of opal and amber tRNA(Lys) suppressors were examined, revealing that U35 and A73 are important determinants of tRNA(Lys) acceptor identity. Several possibilities are discussed for the general significance of having tRNA acceptor identity in the same positions in different tRNA acceptor types, as exemplified by positions 35 and 73 in tRNA(Arg) and tRNA(Lys).