RNAs 3 and 4 of the multicomponent genome of beet necrotic yellow vein virus are dispensable for infection of Chenopodium quinoa leaves. We have used mutagenesis of biologically active RNA 3 transcripts to identify 5'-proximal sequences essential in cis for RNA 3 amplification. One such element, Box I, (nucleotides 283-292) was complementary to the first 10 residues (Box I') following the 5'-terminal cap. A second cis-active element (Box II) was identified between nucleotides 237-244 and was complementary to nucleotides 16-23 (Box II'). Other cis-active sequences exist between Box II' and II but have not been mapped to fine scale. Most sequence substitutions in Boxes I and II or in the 5'-proximal complementary sequences were lethal but compensatory mutations designed to restore Box I/I' or Box II/II' base pairing restored viability, suggesting that secondary structure involving these elements rather than their exact sequence is the critical feature. Transcripts bearing short deletions near residue 200 were replicated but did not assemble into virions, indicating that this region contains or contributes to a cis-active encapsidation signal. Similar experiments with RNA 4 transcript have shown that 5'-proximal cis-essential elements are limited to the first 400 residues of this RNA. Essential subdomains within this region have not been mapped but there are no structures obviously homologous to Boxes I/I' and II/II' of RNA 3.