The recognition of tRNALeu, one of the class II tRNAs having a long variable arm, by leucyl-tRNA synthetase in Saccharomyces cerevisiae was studied using the T7 transcription system. Exchanging the anticodon arm of tRNALeu but not the D- or T psi C-arm to that of tRNASer seriously affected the leucine accepting activity. Two nucleotides in the anticodon loop, A35 and G37, were found to be important for leucylation. It was also found that the discriminator base, A73, is required for leucylation, and G73 of tRNASer functions as a negative identity determinant for leucyl-tRNA synthetase. Introducing a set of three base substitutions at positions 35, 37 and 73 was sufficient to convert tRNASer into an efficient leucine acceptor. These results indicate that the identity elements of tRNALeu lie at the second position of the anticodon and the 3' adjacent to the anticodon as well as the discriminator position. Such a sequence specific recognition manner is significantly different from that of Escherichia coli, in which not the anticodon but the tertiary structural elements play a key role in discriminating from other class II tRNAs. The leucine system is the first example which shows that the requirement of the anticodon sequence is variable among species.