A genomic sequence encoding mitochondrial methionyl-tRNA synthetase (MetRS) was determined from a pathogenic fungi Candida albicans. The gene is distinct from that encoding the cytoplasmic MetRS. The encoded protein consists of 577 amino acids (aa) and contains the class I defining sequences in the N-terminal domain and the conserved anticodon-binding amino acid, Trp, in the C-terminal domain. This protein showed the highest similarity with the mitochondrial MetRSs of Saccharomyces cerevisiae and Shizosaccharomyces pombe. The mitochondrial MetRSs of these fungi were distinguished from their cytoplasmic forms. The protein lacks the zinc binding motif in the N-terminal domain and the C-terminal dimerization appendix that are present in MetRSs of several other species. Escherichia coli tRNAMet was a substrate for the encoded protein as determined by genetic complementation and in vitro aminoacylation reaction. This cross-species aminoacylation activity suggests the conservation of interaction mode between tRNAMet and MetRS.