Biologically active in vitro transcripts were synthesized from a cloned cDNA of a defective RNA (D RNA) of clover yellow mosaic virus (CYMV) and were used to determine if a hexanucleotide motif (5'-ACUUAA) conserved in the 3' noncoding region of potexvirus RNAs is essential for accumulation of progeny D RNA in planta. Deletion of rearrangement of the entire hexanucleotide sequence in the D RNA resulted in no detectable accumulation of progeny D RNA when coinoculated with helper CYMV RNA. Single-base substitutions of the four central nucleotides of the hexanucleotide sequence revealed that viable D RNAs can tolerate single residue changes at positions 3 and 5 only. These results suggest that the hexamer motif is involved in the process of D RNA propagation. The hexanucleotide sequence was also identified in the negative strand of potexvirus RNAs in the regions proposed to represent subgenomic RNA (sgRNA) promoters. In addition, the hexamer motif is present in similar regions in carlavirus RNAs. The conservation of this hexanucleotide (in orientation and position) in potexvirus and carlavirus RNAs, which serve as templates for full-length negative-strand synthesis and sgRNA production, strongly suggests that it plays a functional role in the synthesis of viral RNAs. Taken together, our data support our previous proposal (Bancroft et al., 1991. J. Gen. Virol. 72, 2173-2181) that the hexanucleotide sequence acts as a cis element involved in synthesis of full-length negative-sense viral RNA and further implicate the sequence in a similar role in production of positive-sense sgRNA.