A cDNA of human origin is shown to encode a tRNA isopentenyl transferase (E.C. 2.5.1.8). Expression of the gene in a Saccharomyces cerevisiae mutant lacking the endogenous tRNA isopentenyl transferase MOD5 resulted in functional complementation and reintroduction of isopentenyladenosine into tRNA. The deduced amino acid sequence contains a number of regions conserved in known tRNA isopentenyl transferases. The similarity to the S. cerevisiae MOD5 protein is 53%, and to the Escherichia coli MiaA protein 47%. The human sequence was found to contain a single C2H2 Zn-finger-like motif, which was detected also in the MOD5 protein, and several putative tRNA transferases located by BLAST searches, but not in prokaryotic homologues.