The efficient replication of large DNA viruses requires dNTPs supplied by a viral ribonucleotide reductase. Viral ribonucleotide reductase is an early gene product of both vaccinia and herpes simplex virus. For productive infection, the apoprotein must scavenge iron from the endogenous, labile iron pool(s). The membrane-permeant, intracellular Fe(2+) chelator, 2,2'-bipyridine (bipyridyl, BIP), is known to sequester iron from this pool. We show here that BIP strongly inhibits the replication of both vaccinia and herpes simplex virus, type 1. In a standard plaque assay, 50 microm BIP caused a 50% reduction in plaque-forming units with either virus. Strong inhibition was observed only when BIP was added within 3 h post-infection. This time dependence was observed also in regards to inhibition of viral late protein and DNA synthesis by BIP. BIP did not inhibit the activity of vaccinia ribonucleotide reductase (RR), its synthesis, nor its stability indicating that BIP blocked the activation of the apoprotein. In parallel with its inhibition of vaccinia RR activation, BIP treatment increased the RNA binding activity of the endogenous iron-response protein, IRP1, by 1.9-fold. The data indicate that the diiron prosthetic group in vaccinia RR is assembled from iron taken from the BIP-accessible, labile iron pool that is sampled also by ferritin and the iron-regulated protein found in the cytosol of mammalian cells.