RNA polymerase III promoter is located within the coding region in all eukaryotic tRNA genes, whereas in prokaryotic tRNA genes, the promoter is located upstream of the transcription initiation site. We analyzed the nucleotide sequence context of the A and B boxes of RNA polymerase III promoters from different unicellular eukaryotes, plants, and animals and the homologous sequences in the tRNA genes of prokaryotic species (Archaea, Eubacteria). The long and short sequence variants of the A box are nonrandomly distributed across different types of the eukaryotic tRNA genes. In contrast, the sequences from the prokaryotic counterparts of the long and short variants are randomly scattered among prokaryotic tRNA genes. The sequence diversity of the 3' portion of the A box in the eukaryotic tRNA genes was sharply reduced compared to that of the homologous segments of the prokaryotic tRNA genes. Analysis of the frequencies of oligonucleotide variants from different parts of the A box suggested that the prominent changes in the structure of the A box occurred at about the same time in all tRNA genes during the prokaryote eukaryote evolutionary transition.