In eukaryotic tRNAs, guanosines in position 26 (G26), located at the junction between the D-stem and the anticodon stem of tRNA, are usually modified to N2,N2-dimethylguanosine (m2(2)G). Although G26 is a prerequisite for biosynthesis of m2(2)G26, it is not self-sufficient for the formation of the dimethylated G26, since in exceptional cases eukaryotic tRNAs have an unmodified G26. In the yeast Saccharomyces cerevisiae the only tRNA species with an unmodified G26 is tRNAAsp. Using in vitro transcripts of this tRNA, as well as of yeast tRNAPhe, a tRNA containing m2(2)G26 in vivo, we have investigated the requirements on tRNA sequences and structures for the formation of m2(2)G26 by the yeast enzyme, i.e. in a homologous in vitro system. We have now demonstrated that G26 was efficiently dimethylated in vitro also after deletion of the entire anticodon stem and loop. We conclude that the elements necessary for a productive interaction between G26 in nuclear coded yeast tRNAs and the yeast G26 modifying enzyme are located within the core of the tRNA. For modification of G26 to m2(2)G26 via monomethylated G26, important primary and secondary structural elements in the tRNAs are a size of at least five nucleotides in the variable loop together with two G-C base pairs in the D-stem. This is the first case reported where the minimal requirements on nuclear coded tRNAs for a yeast modifying enzyme has been elucidated.