A series of 29 pyrimidines comprising analogues of 6-azauridine (e.g. 2- and 4-thio-6-azauridine), 6-substituted uridines (including several known inhibitors of orotidine 5'-monophosphate decarboxylase, ODCase, e.g. pyrazofurin), and 6-azauridine-5'-(ethyl methoxyalaninyl) phosphate (a potential prodrug of 6-AU-5'-MP) were synthesized and evaluated in vitro and in vivo against five RNA viruses: Japanese encephalitis (JE), yellow fever (YF), sandfly fever (SF), Punta Toro (PT) and Venezuelan equine encephalomyelitis (VEE) viruses. 2-Thio-6-azauridine demonstrated the best in vitro activity against all five viruses. However, in vivo activity was not observed in JE-, PT- and VEE-infected mice. The phosphate prodrug of 6- azauridine was significantly more effective than the parent compound in the PT virus mouse model. Optimum in vivo dose/route/schedule was determined for pyrazofurin in PT-virus-infected mice.