Polymerization of 4-thiouridine 5′-diphosphate and 4-thiothymidine 5′-diphosphate by polynucleotide phosphorylase from Micrococcus lysodeikticus. 4-Thiouridine 5′-diphosphate (s4UDP) and 4-thiothymidine 5′-diphosphate were polymerized by polynucleotide phosphorylase from Micrococcus lysodeikticus only in the presence of UDP, ADP, IDP, or CDP. The copolymerization of s4UDP and UDP was studied in detail. It was found, that the polymerization was inhibited by increasing amounts of s4UDP. Since UDP polymerization could be inhibited by addition of poly (U1.25, s4U), it is supposed that the copolymerization of s4UDP and UDP is inhibited by polynucleotides poly (U, s4U). 4-Thiouridylyl-(3′-5′)-4-thiouridine and uridylyl-(3′-5′)-4-thiouridine have been found to stimulate the poly U synthesis depending on the primer. Polynucleotides of the general formula poly (U, s4U) were isolated on a preparative scale. Their compositions could be calculated from ultraviolet absorption spectra. © 1969.