Abstract: In the yeast Saccharomyces cerevisiae, uridine ribohydrolase activity is important for recycling, via the salvage pathway, pyrimidine deoxy- and ribonucleosides into uracil required for the growth of strains lacking the de novo pyrimidine synthesis pathway. We have shown that not only uridine and cytidine, but also 5-fluorouridine, 5-fluorocytidine and deoxycytidine are substrates for this enzyme. We identified, cloned and characterized the corresponding URH1 gene and its physiological function was determined by the measurement of metabolic fluxes in several mutants impaired in the pyrimidine salvage pathway. Sequence comparison revealed strong homology between Urh1p and the inosine/uridine-preferring nucleosidase and inosine/adenosine/guanosine nucleoside hydrolase proteins from the parasitic organisms Crithidia fasciculata and Trypanosoma brucei brucei. Moreover, the Asp and His residues in the putative active site were conserved. Site-directed mutagenesis demonstrated that the conserved His residue is involved in catalysis. These results allow us to speculate that the structure and catalytic mechanism of Urh1p are similar to the inosine/uridine nucleoside hydrolase from C. fasciculata.