In the majority of eukaryotic tRNAs, the guanosine at position 26 is modified by a dimethyl group, but so far a function of this modification has not been detected. We isolated the Schizosaccharomyces pombe gene, trm1, encoding the tRNA N2, N2-dimethylguanosine-26 methyltransferase. Strains having the gene deleted completely lack N2,N2-dimethylguanosine. In strains carrying the weak ochre tRNA suppressor sup3-i, deletion of trm1 abolishes suppression indicating that the trm1 deletion acts as an antisuppressor mutation. The result suggests that in vivo N2, N2-dimethylguanosine-26 increases the capacity of the sup3-i serine tRNA to translate the UAA (ochre) codon.