The amino acid sequences of Gcd10p and Gcd14p, the two subunits of the tRNA :(1-methyladenosine-58 ; m 1 A58) methyltransferase (MTase) of Saccharomyces cerevisiae, have been analyzed using iterative sequence database searches and fold recognition programs. The results suggest that the`catalytic' Gcd14p and`substrate binding' Gcd10p are related to each other and to a group of prokaryotic open reading frames, which were previously annotated as hypothetical protein isoaspartate MTases in sequence databases. It is predicted that the prokaryotic proteins are genuine tRNA:m 1 A MTases based on similarity of their predicted active site to the Gcd14p family. In addition to the MTase domain, an additional domain was identified in the N-terminus of all these proteins that may be involved in interaction with tRNA. These results suggest that the eukaryotic tRNA:m 1 A58 MTase is a product of gene duplication and divergent evolution of a possibly homodimeric prokaryotic enzyme. ß