Rhizopus RNase was purified 1000-fold from Glutase with a yield of 2.3 %. It was most active between pH 5 and 5.5. The enzyme appeared not homogeneous in the analytical gel filtration through Sephadex G-100. The enzyme was free from DNase and PDase. Nucleotidases were found, which were inhibited by arsenate. The hydrolytic specificity of the enzyme action was examined by characterization of the digest of yeast RNA. The enzyme produces four kinds of 2’, 3’-cyclic nucleotides, which are slowly hydrolyzed to the corresponding mononucleotides. The cyclic PDase activities showed a preference for cyclic purine nucleotides, and the action for cyclic pyrimidine nucleotides seemed to be slower than that of RNase I. The Rhizopus RNase is useful for preparation of cyclic nucleotides. Further purification is needed for the additional characterization of the enzyme.