pseudouridine was prepared using the ribonuclease-catalyzed reaction described by Heppel et al. (1955). Cytidine 2',3'cyclic phosphate (0.1 mmole) and pseudouridine (100 mg) were dissolved in 0.015 M Tris-chloride buffer (pH 7; 5 ml) and treated with pancreatic ribonuclease (0.1 mg) at 37" for 75 min. The mixture was then shaken with isoamyl alcohol (0.6 ml) and chloroform (0.15 ml) and streaked on Whatman No. 3MM chromatographic paper (66 cm). The components of the mixture were separated with solvent system B and the dinucleoside phosphate band was cut out and eluted (yield, 30 ODU). This product was found to be completely degradable by ribonuclease to cytidine 3'-phosphate and pseudouridine. The dinucleoside phosphate (20 ODU) was dissolved in 0.05 M sodium borate buffer (pH 8.5 ; 0.3 ml) and treated with Cmcp-toluenesulfonate (40 mg). The mixture was allowed to stand for 20 hr. At this time paper chromatography in solvent systems A and B showed that about 70% of the material had been converted to Cmc derivatives. The dinucleoside phosphate and its mono-Cmc derivative had RF values 0.41 and 0.61 (solvent system A); 0.18 and 0.26 (solvent system B), respectively. The mixture was treated with concentrated ammonia for 2 hr and then applied to Whatman No. 3MM chromatographic paper and then separated in solvent system B. The Cmc-dinucleoside phosphate (6 ODU) was dissolved in 0.02 M sodium phosphate buffer (pH 7.0; 0.2 ml) and treated with pancreatic ribonuclease (2.5 pg) at 37" for 4 hr. Paper chromatography in solvent system A showed that about 70% of the Cmc derivative had been converted into a mixture of cytidine 3'-phosphate and Cmc-pseudouridine. With twice the amount of enzyme under the same conditions it was possible to hydrolyze all the dinucleoside phosphate to its components.