Nicotiana benthamiana often displays more intense symptoms after infection by RNA viruses than do other Nicotiana species. Here, we examined the role of RNA-dependent RNA polymerases (RdRPs) in N. benthamiana antiviral defense. cDNAs representing only two genes encoding RdRPs were identified in N. benthamiana. One RdRP was similar in sequence to SDE1/SGS2 required for maintenance of transgene silencing, whereas the second, named NbRdRP1m, was >90% identical in sequence to the salicylic acid (SA)-inducible RdRP from Nicotiana tabacum required for defense against viruses. NbRdRP1m expression was induced by SA treatment or challenge with Tobacco mosaic virus, but the gene and transcript sequences differed from those of other SA-inducible RdRPs in that they contained a 72-nt insert with tandem in-frame stop codons in the 5' portion of the ORF. N. benthamiana plants transformed with an SA-inducible RdRP gene from Medicago truncatula were more resistant to infection by Tobacco mosaic virus, Turnip vein-clearing virus, and Sunn hemp mosaic virus (members of Tobamovirus genus), but not to Cucumber mosaic virus and Potato virus X (members of different genera than the tobamoviruses). Our results indicate that N. benthamiana lacks an active SA- and virus-inducible RdRP and thus is hypersusceptible to viruses normally limited in their accumulation by this RdRP. These findings are significant for those studying virus-induced gene silencing, the hypersensitive response and systemic acquired resistance.