Highly efficient cap-independent translation initiation at the 5′-proximal AUG is facilitated by the 3′ translation enhancer sequence (3′TE) located near the 3′ end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3′TE in regulating viral translation was examined. The 3′TE is required for translation and thus replication of the genomic RNA that lacks a 5′ cap (Allen et al., 1999, Virology 253:139–144). Here we show that the 3′TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3′TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5′ extremity of the sequence required in the 3′ untranslated region (UTR) for cap-independent translation in vivo coincides with the 5′ end of sgRNA2. Thus, sgRNA2 has the 3′TE in its 5′ UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3′TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5′ UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.