The current interest in the role played by divalent cations in the orotate phosphoribosyltransferase (OPRTase)-catalyzed synthesis of orotidine-5'-monophosphate calls for further investigation to gain insight into the nature of the substrate-metal ions-protein interaction. Physical methods such as NMR and ESR spectroscopy have proved to be valuable techniques in achieving this goal, but they need large amounts of protein. As the currently available purification procedures (requiring 9 steps) are rather tedious, we have elaborated a simple and rapid procedure based on affinity chromatography using OMP-Sepharose for the purification of OPRTase from Escherichia coli K-12. The purification process included thermal denaturation, ammonium sulfate fractionation, and affinity chromatography. The purified enzyme had a specific activity of 27 μmol consumed orotate·mg protein⁻¹·min⁻¹, with a yield of approximately 1. It was homogeneous as determined by non-denaturing polyacrylamide gel electrophoresis, and the subunit molecular weight was estimated to be 22,000 (±2000) using SDS gel electrophoresis. The OMP-Sepharose gel was also effective for purifying OPRTase from other organisms, such as baker's yeast. The gel proved to be well suited for OPRTase purification, with evidence of true biospecific fixation of the enzyme to the immobilized ligand rather than non-specific hydrophobic bonding. Elution with orotate or PRPP in the presence of Mg²⁺ demonstrated good affinity of both ligands for the OMP-protein complex. This method provides an efficient means to obtain pure OPRTase for studying its structure and function.