Using an Agrobacterium-mediated transient assay, we screened the 15.5-kb genome of the Beet yellows virus for proteins with RNA silencing suppressor activity. Among eight proteins tested, only a 21-kDa protein (p21) was able to suppress double-stranded (ds) RNA-induced silencing of the green fluorescent protein (GFP) mRNA. Restoration of GFP expression by p21 under these conditions had no apparent effect on accumulation of the small interfering RNAs. In addition, p21 elevated the transient expression level of the GFP mRNA in the absence of dsRNA inducer. Similar activities were detected using homologs of p21 encoded by other members of the genus Closterovirus. Computer analysis indicated that p21-like proteins constitute a novel protein family that is unrelated to other recognized suppressors of RNA silencing. Examination of the subcellular distribution in BYV-infected plants revealed that p21 is partitioned between soluble cytoplasmic form and proteinaceous inclusion bodies at the cell periphery.