We have investigated the mechanism by which ribavirin (Rbv), mycophenolic acid, and 2-amino-1,3,4-thiadiazole inhibit the replication of Sindbis virus in Aedes albopictus cells. In each case there was a good correlation between inhibition of virus replication and a reduction (80–90%) in the level of cellular GTP. The antiviral effects of all three compounds could be reversed by (1) equimolar amounts of xanthosine but not by guanosine, (2) actinomycin D (0.2 μg/ml), and (3) α-amanitin (10 μg/ml) in α-amanitin-sensitive cells but not in α-amanitin-resistant cells. In the case of actinomycin D the reversal of the antiviral effects was correlated with a restoration of the GTP pool to near normal levels and a decrease in the amount of phosphorylated Rbv in acid-soluble cell extracts. The role of the phosphorylated forms of Rbv in inhibiting virus replication and the relationship to cellular RNA synthesis are discussed.