To characterize the substrate specificity of the putative RNA:pseudouridine (Psi)-synthase encoded by the Saccharomyces cerevisiae open reading frame (ORF) YGR169c, the corresponding gene was deleted in yeast, and the consequences of the deletion on tRNA and small nuclear RNA modification were tested. The resulting DeltaYGR169c strain showed no detectable growth phenotype, and the only difference in Psi formation in stable cellular RNAs was the absence of Psi at position 31 in cytoplasmic and mitochondrial tRNAs. Complementation of the DeltaYGR169c strain by a plasmid bearing the wild-type YGR169c ORF restored Psi(31) formation in tRNA, whereas a point mutation of the enzyme active site (Asp(168)-->Ala) abolished tRNA:Psi(31)-synthase activity. Moreover, recombinant His(6)-tagged Ygr169 protein produced in Escherichia coli was capable of forming Psi(31) in vitro using tRNAs extracted from the DeltaYGR169c yeast cells as substrates. These results demonstrate that the protein encoded by the S. cerevisiae ORF YGR169c is the Psi-synthase responsible for modification of cytoplasmic and mitochondrial tRNAs at position 31. Because this is the sixth RNA:Psi-synthase characterized thus far in yeast, we propose to rename the corresponding gene PUS6 and the expressed protein Pus6p. Finally, the cellular localization of the green fluorescent protein-tagged Pus6p was studied by functional tests and direct fluorescence microscopy.