Tetragonia expansa plants infected with a California isolate of beet yellows virus (BYV-60) contained multiple BYV-specific RNAs identified by Northern blot hybridization. These RNAs were identified by cDNA probes specific to six open reading frames (ORFs). One genomic RNA and five subgenomic (sg) RNAs representing the p65/p6.4, p64, p24, p22, and p21 ORFs were identified. A probe derived from the 3′-terminal ORF (p21) hybridized to each of the sgRNAs, indicating the RNAs are 3′ coterminal. Hybridization with 5′- and 3′-end probes indicated that preparations of BYV particles contained the genomic RNA as well as two additional RNA molecules corresponding in size to the coat protein (CP) sgRNA and an unidentified RNA. A Chenopodium quinoa protoplast system also was used to study BYV replication. The temporal accumulation of BYV-specific RNAs and CP was investigated in protoplasts transfected with purified virion RNA. Accumulation of genomic plus-strand RNA was evident as early as 15 h postinoculation. The development of this protoplast system is significant for studies of closterovirus replication.