Using blue Sepharose affinity chromatography, we purified orotidine-5'-phosphate decarboxylase over 600-fold, to near homogeneity, from strains of Escherichia coli harboring the cloned pyrF gene on the multicopy plasmid pDK26. The purified enzyme has a subunit molecular weight of 27,000 but appears to be catalytically active as a dimer. In contrast to yeast enzymes, orotidine-5'-phosphate decarboxylase from E. coli is unstable at pH 6.0. The specific activity and Km values were 220 U/mg and 6 microM, respectively.