Most eukaryotic mRNAs are translated by a "scanning ribosome" mechanism. We have found that unlike the type member of the genus Tobamovirus, translation of the 3'-proximal coat protein (CP) gene of a crucifer infecting tobamovirus (crTMV) (Dorokhov et al., 1993; 1994) occurred in vitro by an internal ribosome entry mechanism. Three types of synthetic dicistronic RNA transcripts were constructed and translated in vitro: (i) "MP-CP-3'NTR" transcripts contained movement protein (MP) gene, CP gene and the 3'-nontranslated region of crTMV RNA. These constructs were structurally equivalent to dicistronic subgenomic RNAs produced by tobamoviruses in vivo. (ii) "deltaNPT-CP" transcripts contained partially truncated neomycin phosphotransferase I gene and CP gene. (iii) "CP-GUS" transcripts contained the first CP gene and the gene of Escherichia coli beta-glucuronidase (GUS) at the 3'-proximal position. The results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained an internal ribosome entry site (IRES(CP)) promoting internal initiation of translation in vitro. Dicistronic IRES(CP), containing chimeric mRNAs with the 5'-terminal stem-loop structure preventing translation of the first gene (MP, deltaNPT, or CP), expressed the CP or GUS genes despite their 3'-proximal localization. The capacity of crTMV IRES(CP) for mediating internal translation distinguishes this CP tobamovirus from the well-known-type member of the genus, TMV UI. The equivalent 148-nt sequence from TMV RNA was incapable of mediating internal translation. Two mutants were used to study structural elements of IRES(CP). It was concluded that integrity of IRES(CP) was essential for internal initiation. The crTMV provides a new example of internal initiation of translation, which is markedly distinct from IRESs shown for picornaviruses and other viral and eukaryotic mRNAs.