Infection of tobacco protoplasts or leaf tissues with peanut stunt virus (PSV) subgroup II strains induced the production of unusual cytoplasmic ribbon-like inclusions. The inclusion structures appeared as long, thin, densely staining sheets that were prevalent within the cytoplasm, accumulating most commonly near vacuoles. Numerous virions and ribosomes could be seen adjacent to the inclusion surfaces. The formation of these novel inclusions appeared to be subgroup specific, since infection of tobacco with PSV strains W and B (subgroup II), but not strains ER, V, and J (subgroup I), induced the inclusions. Furthermore, inclusion formation was shown to be host specific, because the inclusions were not detected in either of two leguminous host species infected with PSV subgroup II strains. Using tobacco protoplasts electroporated with various assortments of infectious RNA transcripts derived from cDNA clones of genomic RNAs of PSV-ER and PSV-W, we demonstrated that induction of the unusual ribbon-like inclusions maps to PSV-W (subgroup II) RNA3. This conclusion is consistent with the finding that PSV strain BV-15, a natural intraspecific reassortant that derives its RNA2 and RNA3 from a subgroup I strain, did not induce inclusion formation.