Uridine phosphorylase from Escherichia coli has been purified to homogeneity. The enzyme was found to have a molecular weight of 176000 and to consist of 8 probably identical subunits with molecular weights of 22000. These numbers were determined from equilibrium centrifugations in the analytical ultracentrifuge, from dodecylsulphate gel electrophoresis and from amino acid analysis. Moreover the following physico-chemical constants were determined: s020,w = 8.2 x 10(-13) s, upsilon2 = 0.751 cm3/g, A1%280 (1 cm) = 6.73 and a specific activity of 183 units/mg towards uridine. The enzyme shows some activity towards deoxyuridine and thymidine. The activity is not impaired through substitution by bromo, fluoro or methyl groups in the 5-position of the uracil base, but no enzymatic activity is observed when cytosine base is used in the nucleoside substrate.