To investigate the sequence requirements of Escherichia coli tRNA(Ser) for recognition by seryl-tRNA synthetase, various mutants of unmodified tRNA(Ser) were constructed. Substitution of G2.C71 by C2.G71, but not by A2.U71 or U2.A71, impaired the serine-accepting activity, indicating that this position is not involved in recognition by seryl-tRNA synthetase, but contributes to discrimination from other tRNAs processing C2.G71 such as tRNA(Leu). Other nucleotides characteristic of tRNA(Ser), including the discriminator base, were not involved in recognition by seryl-tRNA synthetase. The anticodon was not involved, as suggested by its sequence variety within the isoacceptors. The long variable arm composed of over ten nucleotides, which is a characteristic feature of tRNA(Ser) together with tRNA(Leu) and tRNA(Tyr), was stem-length-specifically, but not sequence-specifically, important for recognition. In order to introduce a sufficient serine-accepting activity to a tRNA(1LEU) transcript in vitro, besides the change from C2.G71 to G2.C71, the following elements had to be changed to those characteristic of tRNA(Ser): the sequence in the D-loop, the stem pairing pattern of the variable arm, the tertiary base-pair 15.48 and the nucleotide at position 59 in the T psi C-loop. None of the nucleotides at these changed positions was involved in base-specific recognition, indicating that seryl-tRNA synthetase selectively recognizes tRNA(Ser) on the basis of its characteristic tertiary structure rather than the nucleotides specific to tRNA(Ser).