The protein products of two yeast Saccharomyces cerevisiae genes (YNL292w and CBF5) display a remarkable sequence homology with Escherichia coli tRNA:pseudouridine-55 synthase (encoded by gene truB). The gene YNL292w coding for one of these proteins was cloned in an E.coli expression vector downstream of a His6-tag. The resulting recombinant protein (Pus4) was expressed at high level and purified to homogeneity by metal affinity chromatography on Ni2+-NTA-agarose, followed by ion-exchange chromatography on MonoQ. The purified Pus4p catalyzes the formation of pseudouridine-55 in T7 in vitro transcripts of several yeast tRNA genes. In contrast to the known yeast pseudouridine synthase (Pus1) of broad specificity, no other uridines in tRNA molecules are modified by the cloned recombinant tRNA:Psi55 synthase. The disruption of the corresponding gene YNL292w in yeast, which has no significant effect on the growth of yeast cells, leads to the complete disappearance of the Psi55 formation activity in a cell-free extract. These results allow the formal identification of the protein encoded by the yeast ORF YNL292w as the only enzyme responsible for the formation of Psi55 which is almost universally conserved in tRNAs. The substrate specificity of the purified YNL292w-encoded recombinant protein was shown to be similar to that of the native protein present in yeast cell extract. Chemical mapping of pseudouridine residues in both cytoplasmic and mitochondrial tRNAs from the yeast strain carrying the disrupted gene reveals that the same gene product is responsible for Psi55 formation in tRNAs of both cellular compartments.