A procedure is described for the synthesis of the title compounds via phosphotriester intermediates. The 2-cyanoethyl group is used to protect the P-SH function during the course of the synthesis. Resolution of the phosphorus-diastereomers is accomplished at the phosphotriester stage. Removal of the 2-cyanoethyl group without racemization at phosphorus, followed by removal of the other protective groups affords the optically pure diastereomers of uridyl (3′-5′) adenyl O,O-phosphorothioate. Their designation as Rp and Sp follows from the stereospecificity of the hydrolysis cataSyzed by RNase A.