The plus-strand RNA genome of Tobacco necrosis virus-D (TNV-D) expresses its polymerase via translational readthrough. The RNA signals involved in this readthrough process were characterized in vitro using a wheat germ extract translation system and in vivo via protoplast infections. The results indicate that (i) TNV-D requires a long-range RNA-RNA interaction between an extended stem-loop (SL) structure proximal to the readthrough site and a sequence in the 3'-untranslated region of its genome; (ii) stability of the extended SL structure is important for its function; (iii) TNV-D readthrough elements are compatible with UAG and UGA, but not UAA; (iv) a readthrough defect can be rescued by a heterologous readthrough element in vitro, but not in vivo; and (v) readthrough elements can also mediate translational frameshifting. These results provide new information on determinants of readthrough in TNV-D and further support the concept of a common general mechanism for readthrough in Tombusviridae.