Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35-40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 mu M periodate oxidized enzyme are allowed to react with 1 mi (packed volume) of AE-Bio-Gel P-2 at 6 +/- 1 degrees C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K-m but resulted in a 2.5-fold decrease in the V-max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2＇,3＇ cyclic nucleotides in 14 h, at pH 5.5 and 37 degrees C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity.