Uridine nucleosidase (EC 3.2.2.3) was purified from commercial bakers' yeast to homogeneity, as judged by a single band observed on polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme, estimated by gel filtration, was approximately 32,500. Polyacrylamide electrophoresis in 0.2% sodium dodecyl sulfate showed the presence of two apparently identical subunits of 17,000 molecular weight. The amino acid composition indicated a large excess of glutamic acid and aspartic acid over other amino acid residues and a very low content of tyrosine and tryptophan. Th SH groups analysis performed with 5,5'-dithiobis (2-nitrobenzoic acid) on thenative protein as well as in the presence of 1% sodium dodecyl sulfate showed the existence of one sulfhydryl group per mole of enzyme. Uridine nucleosidase is active on uridine and 5-methyluridine (ribosylthymine) resulting inactive toward all other pyrimidine and purine nucleosides tested. The Km values for uridine and 5-methyluridine were 0.86 x 10(-3) M and 1.66x10--3M, respectively. The optimal pH is around 7.0. The isoelectric point is 5.1. Among a variety of compounds tested only ribose and glucose 6-phosphate were inhibitory and Ki values were 7.2 mM and 0.19 mM, respectively. Furthermore, ribosylthymine competitively inhibited the hydrolysis of uridine. The type of all inhibitions was competitive and the n' values of the Hill plots were near 1. The effect of temperature on the enzyme activity plotted accoring to Arrhenius gave a value of E = 4740 cal per mole. The enzyme in 100 mM phosphate, pH = 7.0, is stable at 4 degrees for 15 days without any loss of activity.