Previously, we showed that these tRNA Ser CAGs would have originated from the serine tRNA corresponding to codon UCG . This suggests an evolutionary pathway in which conversion from A to m 1 G would have taken place at position 37 just after the emergence of tRNA Ser CAG had brought about a change in the universal code. Since such a mutation at position 37 might potentially result in the leucylation of tRNA Ser CAG, we attempted to elucidate the charging properties of these tRNA Ser CAGs both in vitro and in vivo. Based on the results of in vitro aminoacylation reactions using tRNA variants constructed by the microsurgery method, the direct analysis of aminoacylated tRNAs in cells and a genetic approach, we demonstrate here that these serine tRNAs are actually leucylated both in vitro and in vivo. Furthermore, m 1 G at position 37 was found to be indispensable for the leucylation of tRNA Ser CAGs. In fact, the tRNA Ser CAG of C.cylindracea, which has A at position 37, exhibits no leucylation activity. C.cylindracea has a high GϩC content (63%) and utilizes CUG as a major serine codon. However, the other Candida species have no such high GϩC content and utilize the CUG as a minor serine codon (Kawaguchi et al., 1989; Lloyd and Sharp, 1992; our unpublished observation) . Considering the relationship between the usage of the codon CUG as serine and the leucylation properties of tRNA Ser CAG, it seems that only Candida species with a genome in which the incidence of the CUG serine codon is very low possess serine tRNA Ser CAG that can be leucylated. Furthermore, such tRNA Ser CAGs charged with heterogeneous amino acids should be utilized equally in the translation process. This is the first demonstration that a single tRNA species is assigned to two different amino acids in the cell. We propose designating this type of codon having multiple amino acid assignment as a 'polysemous codon'. The correlation between the dual-assignment state and the pathway of genetic code diversification is also discussed. 