Several plant virus mutants, in which genes encoding silencing suppressor proteins have been deleted, are known to induce systemic or localized RNA silencing against themselves and other RNA molecules containing homologous sequences. Thus, it is thought that many cases of cross-protection, in which infection with a mild or asymptomatic virus mutant protects plants against challenge infection with closely related virulent viruses, can be explained by RNA silencing. We found that a cucumber mosaic virus (CMV) mutant of the subgroup IA strain Fny (Fny-CMVDelta2b), which cannot express the 2b silencing suppressor protein, cross-protects tobacco (Nicotiana tabacum) and Nicotiana benthamiana plants against disease induction by wild-type Fny-CMV. However, protection is most effective only if inoculation with Fny-CMVDelta2b and challenge inoculation with wild-type CMV occurs on the same leaf. Unexpectedly, Fny-CMVDelta2b also protected plants against infection with TC-CMV, a subgroup II strain that is not closely related to Fny-CMV. Additionally, in situ hybridization revealed that Fny-CMVDelta2b and Fny-CMV can co-exist in the same tissues but these tissues contain zones of Fny-CMVDelta2b-infected host cells from which Fny-CMV appears to be excluded. Taken together, it appears unlikely that cross-protection by Fny-CMVDelta2b occurs by induction of systemic RNA silencing against itself and homologous RNA sequences in wild-type CMV. It is more likely that protection occurs through either induction of very highly localized RNA silencing, or by competition between strains for host cells or resources.