Background:The antipoxviral activities and phosphorylation of N-methanocarbathymidine ([ N]-MCT) and four 5-halo-2'-deoxyuridines, namely 5-fluoro- (FdU), 5-chloro- (CldU), 5-bromo- (BrdU), and 5-iodo- (IdU) derivatives, were explored.Methods:Antiviral activities and nucleoside metabolism were determined in C127I mouse, LLC-MK2monkey, and A549 human cells infected with thymidine-kinase-containing and -deficient (TK+and TK−) vaccinia (WR strain) viruses.Results:The antiviral potencies of CldU, BrdU and IdU were increased 16–26-fold in LLC-MK2cells infected with TK+compared with TK−virus infections, but enhancement of activity was much less in the other cell lines. ( N)-MCT was nearly equally active against TK+and TK−viruses in the three cell lines. Antiviral activity of FdU was associated with cytotoxicity. Uninfected and infected cells metabolized compounds to mono-, di- and triphosphates. The thymidine, BrdU and IdU triphosphate levels were higher in C127I and LLC-MK2cells infected with TK+than with TK−virus. ( N)-MCT monophosphate levels were much higher in TK+virus-infected cells, but without corresponding increases in ( N)-MCT triphosphate. Furthermore, TK+virus infections did not appreciably alter ( N)-MCT triphosphate levels in other mouse (L929), monkey (MA-104 and Vero) and human cell lines (A549). Antiviral potency of the compounds was greater in C127I than in LLC-MK2cells, yet lower intracellular triphosphate levels were found in C127I cells.Conclusion:We conclude that viral TK plays an important role in increasing the antiviral potencies of these compounds in some cell lines, but minimally in others. These findings may have implications in treating infected animals with compounds that are dependent upon poxvirus TK for their activation, because viral TK activity may vary greatly due to cell type.