Cytoplasmic and mitochondrial tRNAs contain several pseudouridylation sites, and the tRNA:-synthase acting at position 32 had not been identified in Saccha-romyces cerevisiae. By combining genetic and biochemical analyses, we demonstrate that two enzymes, Rib2/ Pus8p and Pus9p, are required for 32 formation in cytoplasmic and mitochondrial tRNAs, respectively. Pus9p acts mostly in mitochondria, and Rib2/Pus8p is strictly cytoplasmic. This is the first case reported so far of two distinct tRNA modification enzymes acting at the same position but present in two different compartments. This peculiarity may be the consequence of a gene fusion that occurred during yeast evolution. Indeed , Rib2/Pus8p displays two distinct catalytic activities involved in completely unrelated metabolism: its C-terminal domain has a DRAP-deaminase activity required for riboflavin biogenesis in the cytoplasm, whereas its N-terminal domain carries the tRNA:32-synthase activity. Pus9p has only a tRNA:32-synthase activity and contains a characteristic mitochondrial targeting sequence at its N terminus. These results are discussed in terms of RNA:-synthase evolution.