To evaluate the extent to which brome mosaic virus (BMV) coat protein (CP) gene is involved in the process of cell-to-cell movement. Chenopodium quinoa plants were coinoculated with BMV wild-type RNAs 1 and 2 and a RNA3 variant containing either the beta-glucuronidase (GUS) gene in place of CP (B3/CP-GUS), to be subjected to GUS analysis, or a deletion in the CP gene to be analyzed by fluorescence in situ hybridization (FISH). Irrespective of time postinoculation, GUS expressed from the subgenomic mRNA of B3/CP-GUS was restricted to initially infected, single epidermal cells. The defective cell-to-cell movement exhibited by B3/CP-GUS was not complemented in trans when transcripts of B3 delta MP, a RNA3 variant capable of synthesizing functional wt CP but not movement protein (MP), were added to the inoculum. Application of FISH, a technique versatile in discriminating subliminal infections from efficient cell-to-cell spread in nonpermissive and permissive hosts, respectively, to leaves inoculated with BMV RNA3 variants defective in CP synthesis, confirmed that the resulting infections were subliminal. These data provide direct evidence for the requirement of an encapsidation-competent CP to be expressed in conjunction with a functional MP for efficient cell-to-cell spread of BMV.