Using a genetic screen in yeast aimed at identifying cellular factors involved in initiator and elongator methionine tRNA discrimination in the translational process, we have identified a mutation that abolish the requirement for elongator methionine tRNA. The gene affected, which we call the ribosylation of the initiator tRNA gene or RIT1, encodes a 2'-O-ribosyl phosphate transferase. This enzyme modifies exclusively the initiator tRNA in position 64 using 5'-phosphoribosyl-1'-pyrophosphate as the modification donor. As the initiator tRNA participates both in the initiation and elongation of translation in a rit1 strain, we conclude that the 2'-O-ribosyl phosphate modification discriminates the initiator tRNAs from the elongator tRNAs during protein synthesis. The modification enzyme was shown to recognize the stem-loop IV region that is unique in eukaryotic cytoplasmic initiator tRNAs.