Abstract: The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNAMet(i)) by the addition of a 2'-O-ribosyl phosphate group to Adenosine 64. As a result, tRNAMet(i) is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification is not essential for the function of tRNAMet(i) in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1alpha (eEF-1alpha), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1alpha are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNAMet(i) for elongation. Thus, under conditions in which the components of the ternary met-tRNAMet(i):GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2'-O-ribosyl phosphate modification in tRNAMet(i) is important for the provision of adequate amounts of tRNAMet(i) for formation of this ternary complex.