Barley yellow dwarf virus (BYDV) is an important pathogen of cereal crops. It has a single-stranded positive sense genomic RNA (gRNA), 5.7 kilobases. During infection of a plant cell, BYDV generates a nested set of three subgenomic mRNAs (sgRNAs) for expression of its 3'-proximal genes. The goal of this study was to map and characterize cis-acting RNA signals involved in transcription and replication of BYDV RNA. Three sgRNA promoters and the 3' origin of replication were characterized. The sgRNA1 promoter was mapped to a 98 nt region that contains two stem-loop structures. A combination of primary and secondary structural elements was required for promoter activity. Most of the promoter sequence (75 nt) is located upstream of the transcription initiation site. In contrast, sgRNA2 and sgRNA3 promoters contained the majority of their sequences downstream of the sgRNA start sites. SgRNA2 promoter was mapped to a 143 nt region predicted to fold into a cloverleaf-like structure. SgRNA3 promoter is 44 nt long and was predicted to form a hairpin and a single-stranded RNA region. Sequence and structure comparisons of the three subgenomic promoters did not reveal any similarities indicating that transcription of BYDV sgRNAs is controlled by extremely different cis-elements;The 3' origin of replication of BYDV capable of supporting a basal level of replication was mapped to the 104 3' terminal nucleotides. Based on the computer prediction, phylogenetic and mutational analysis, and nuclease sensitivity assays, this region forms four stable stem-loop structures (SL1-SL4, 3' to 5) that contain terminal GNRA and UNCG tetraloops. Each stem-loop was indispensable for virus replication according to results of deletion mutagenesis. Loop sequences of SL1 and SL2 were not important for replication, whereas loops of SU and SU preferred the GNRA consensus sequence. RNA secondary structure and not the primary sequence of the stems in SL1 through SL4 is important for viral RNA replication in oat protoplasts;Characterization of the cis-acting elements required for transcription and replication of BYDV is an important step towards better understanding of the basic mechanisms of the viral life cycle, which may help in development of new antiviral strategies.