Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus and is the prototype member of the genus Tombusvirus. The genomes of members of this genus are not polyadenylated, and prevailing evidence supports the absence of a 5' cap structure. Previously, a 167-nucleotide-long segment (region 3.5) located near the 3' terminus of the TBSV genome was implicated as a determinant of translational efficiency (S.K. Oster, B. Wu and K. A. White, J. Virol. 72:5845-5851, 1998). In the present report, we provide evidence that a 3'-proximal segment of the genome, which includes region 3.5, is involved in facilitating cap-independent translation. Our results indicate that (i) a 5' cap structure can substitute functionally for the absence of region 3.5 in viral and chimeric reporter mRNAs in vivo; (ii) deletion of region 3.5 from viral and chimeric mRNAs has no appreciable effect on message stability; (iii) region 3.5 represents part of a larger 3' proximal element, designated as the 3' cap-independent translational enhancer (3'CITE), that is required for proficient cap-independent translation; (iv) the 3'CITE also facilitates cap-dependent translation; (v) none of the major viral proteins are required for 3'CITE activity; and (vi) no significant 3'CITE-dependent stimulation of translation was observed when mRNAs were tested in vitro in wheat germ extract under various assay conditions. This latter property distinguishes the 3'CITE from other characterized plant viral 3'-proximal cap-independent translational enhancers. Additionally, because the 3'CITE overlaps with cis-acting replication signals, it could potentially participate in regulating the initiation of genome replication.