The antiviral activity of the 5-thiocyanatopyrimidine nucleosides 5-NCSrU ∗ ∗ Abbreviations: 5-NCSrU, 5-thiocyanatouridine; 5-NCSdU, 5-thiocyanato-2'-deoxyuridine: 5-NCSaraU, 5-thiocyanato uracil arabinoside or 1-β- d-arabinofuranosyl-5-thiocyanatouracil; tri-O'-acetyl-5-NCSrU, 2',3',5'-tri- O-acetyl-5-thiocyanatouridine; ara-C, cytosine arabinoside, 1-β- d-arabinofuranosylcytosine; 5-ethyl-dU, 5-ethyl-2'-deoxyuridine; dT, 2'-deoxythymidine; 5-IdU, 5-iodo-2'-deoxyuridine; CCID 50, cell culture infecting dose 50 (dose infecting 50% of the cell cultures); PFU, plaque forming units. , 5-NCSdU, 5-NCSaraU and tri- O′-acetyl-5-NCSrU has been evaluated in primary rabbit kidney (PRK) cell cultures challenged with either DNA (vaccinia, herpes simplex) or RNA (vesicular stomatitis) viruses. 5-NCSdU inhibited vaccinia virus multiplication at 10 μg/ml, and vaccinia and herpes simplex virus induced cytopathogenicity at 4 μg/ml. Tri- O′-acetyl-5-NCSrU inhibited vesicular stomatitis virus-induced cytopathogenicity at 1–10 μg/ml. None of the compounds had profound effects on host cell RNA or DNA synthesis, even at 200 μg/ml, as monitored by [ 3H]uridine and [ 3H]thymidine incorporation respectively, except 5-NCSdU, which brought about a 10–30-fold increase of [ 3H]thymidine incorporation at 200 μg/ml. The inhibitory effect of 5-NCSdU on vaccinia virus replication and its stimulatory effect on [ 3H]thymidine incorporation were almost completely reversed by thymidine at concentrations 100 times lower than that of the thiocyanato derivative. When treated with dithiothreitol, the 5-thiocyanatopyrimidine nucleosides also lost a significant part of their biological activity, presumably due to reduction to the corresponding 5-mercapto analogs.