SUMMARY Citrus tristeza virus (CTV) has within the 3'-half of the genome ten open reading frames (ORFs) that are expressed through a series of 3'-coterminal subgenomic (sg) messenger (m)RNAs that, in general, function as monocistronic mRNAs with only the 5'-most ORF translated. Yet only nine sg mRNAs have been detected, suggesting that ORF 3, which is predicted to encode a small hydrophobic protein of approximately 6 kDa (p6), might be expressed in some other manner, perhaps from a functionally dicistronic sg mRNA. However, when we positioned the p6 gene near the 3'-terminus of a minimal CTV replicon to amplify greatly the level of production of the putative sg mRNA, we found a minimal level of a p6 mRNA, thus providing evidence for a separate mRNA for each 3' ORF. The 5' termini of the sg mRNAs and the cis-acting elements (controller elements-CEs) that regulate the production of the p6 gene and the adjacent HSP70h (heat shock protein 70 homologue) were located further upstream of the ORFs compared with the other CTV CEs. Both preferentially initiated synthesis with an adenylate, as has been shown for the more highly expressed 3' genes; but in contrast, the p6 sg mRNA occasionally initiated with a guanylate. Although the nucleotide sequences and the computer-predicted secondary structures of the HSP70h CE were similar to those previously described for other 3' CEs, those of the p6 CE were quite different, suggesting that CE strength is related to proximity to an ideal CE conformation. The lack of similarity between different CTV CEs led us to examine how well the CTV replicase complex could initiate sg mRNAs from CEs from different members of the family Closteroviridae. We found that the CTV replicase complex efficiently initiated production of sgRNAs from the p6 and HSP70h CEs from Beet yellows virus, with the HSP70h CE initiating at the same nucleotide as within the homologous virus, indicating that the mode of recognition of the CEs is similar. However, CEs from a more distantly related member of the Closteroviridae, Lettuce infectious yellows virus, did not function to produce sg mRNAs in CTV.