Uterine fluid was collected from estrogen-primed rats which had each previously been subjected to an operation to close the cervical junction of the uterus. Starting from the uterine fluid, uterine fluid peroxidase was purified by carboxymethyl cellulose column chromatography followed by gel filtration on Sephacryl S-200. The purity of the enzyme preparation, as estimated by microelectrophoresis on polyacrylamide gel, was found to be 70-95%. Absorption spectra of the peroxidase and its derivatives resembled those of lactoperoxidase. The pyridine hemochromogen spectrum of the enzyme was also very similar to that of lactoperoxidase. The apparent molecular weight of the enzyme was estimated to be 90,000, being similar to that of lactoperoxidase (78,000). Uterine fluid peroxidase has, however, distinctly different properties from lactoperoxidase, for example, in substrate specificity, pH optimum, inhibition by excess hydrogen peroxide, and isoelectric point.