African green monkey CV-1 cells infected with simian virus 40 (SV40) were incubated in the presence of sodium [35S]thiophosphate. The viral DNA was prepared and the newly synthesized SV40 DNA molecules containing phosphorothioate groups were selectively recovered by affinity chromatography on organomercurial-Sepharose columns. The thio-derivatized viral DNA occurred in both superhelical (form I) and relaxed circular (form II) configurations, and it gave the expected cleavage products upon digestion with the restriction endonucleases Kpn I and HindIII. Enzymatic digestion of 35S-labeled SV40 DNA to its component nucleotides and chromatographic separation of the resultant 5'-labeled phosphorothioate esters established the presence of the label in all four major nucleotides, but mainly in thymidylic acid. The ability to label and recovery selectively the newly synthesized polynucleotides by incubating cells with inorganic thiophosphate complements earlier findings [Sun, I. Y.-C. & Allfrey, V. G. (1982) J. Biol. Chem, 257, 1347--1353] that thiophosphate enters intracellular nucleotide pools and serves as an effective precursor for the phosphorylation of nuclear proteins in vivo.