RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs. In this paper we demonstrate that the purified recombinant Pus1, expressed in Escherichia coli, contains one atom of zinc per 63-kDa monomer, as determined by atomic absorption spectroscopy. This zinc ion could not be removed by treatment with EDTA or urea. However, a zinc-depleted enzyme was obtained after prolonged dialysis against the specific chelating agent 1,10-phenanthroline. Removal of the zinc ion resulted in inactivation of the enzyme with concomitant loss of its ability to bind tRNA. Dialysis of the zinc-depleted inactive enzyme against buffer containing zinc ions led to recovery of up to 25% of bound zinc in parallel with 25% of its initial activity. Removal of the tightly bound zinc atom resulted in a conformational change of the protein, as determined by analytical ultracentrifugation, with minor changes in the internal structure of the protein, as evidenced by circular dichroism and infrared and fluorescence spectroscopy. Our results are consistent with a structural role for the zinc in the tRNA-pseudouridine synthetase Pus1; zinc ion could maintain the association between domains structurally organized around the coordinated metal ion. Zinc chelation was never demonstrated for any of the tRNA-pseudouridine synthetases characterized so far.