6-Azauridine (AzUrd) is a broad-spectrum antimetabolite that inhibits both DNA and RNA virus multiplication. Prior work indicated that several AzUrd-sensitive viruses induced an increase in the level of uridine kinase, and this might explain the selective activity of AzUrd on such viruses. Present studies compared AzUrd sensitive and resistant viruses with respect to their orotic acid pathways by labeling cells with [14C]-orotic acid during the latent period of viral infection. No differences were detected by this method with either vaccinia, Newcastle disease, or vesicular stomatitis viruses. AzUrd inhibits transport of orotic acid into the cell by 30%, while incorporation of orotic acid into cellular RNA is inhibited by 50% (taking into consideration the 30% already noted) when the highest concentration of antimetabolite is used. This suggests that, in addition to blocking orotidylic acid decarboxylase, AzUrd may act on some other site (sites) of action in the inhibition of virus multiplication.