Summary A comparative study of the effects of four pyrimidine nucleoside analogs on the replication of vaccinia virus in HeLa cell cultures was carried out. It was found that 5-iodo-2′-deoxyuridine and 5-trifluoromethyl-2′-deoxyuridine were potent irreversible inhibitors, with selective toxicity to the virus as compared with the cells. Cytosine arabinoside and 5-fluoro-2′-deoxyuridine had no selective toxicity against the virus, and the inhibition produced by the latter compound was transient. When given simultaneously, thymidine reversed the inhibitory effects of 5-fluoro-2′-deoxyuridine, 5-trifluoromethyl-2′-deoxyuridine, and 5-iodo-2′-deoxyuridine; and deoxycytidine reversed the inhibition produced by cytosine arabinoside. If the infected cells were treated with the drugs for 1 day, washed, and normal metabolites added, complete virus production was rescued by thymidine in the case of 5-fluoro-2′-deoxyuridine, partial rescue by deoxycytidine from cytosine arabinoside, but no rescue above the level of the input multiplicity was achieved by thymidine from the inhibition produced by 5-iodo-2′-deoxyuridine and 5-trinuoromethyl-2′-deoxyuridine. These results are discussed in terms of the mechanisms of action of these analogs, and it is concluded that the major (but perhaps not the only) antiviral activity of the latter two compounds is probably a consequence of their incorporation into the viral DNA.