Zta, a transcription factor encoded by Epstein-Barr virus, is efficiently translated from a BRLF1-BZLF1 bicistronic mRNA. In this study, we demonstrate that inserting a stem-loop structure, which is known to block ribosome scanning, in the 5' region of the intercistronic region does not prevent the translation of a luciferase reporter protein from the bicistronic mRNA fused with the firefly luciferase gene, suggesting that the translation does not involve translation reinitiation. Mutational analyses reveal that the region between nucleotides 86 and 125 (region I) of the intercistronic region is essential for the translation. Meanwhile, the region between nucleotides 126 and 165 (region II) is also important since, without this region, the translation is inefficient. The region I sequence is partially complementary to the sequence between nucleotides 1489 and 1524 of 18S rRNA. This homology is significant, since disrupting the homology reduces the translation efficiency. Furthermore, luciferase is efficiently translated if the entire intercistronic region is replaced with a sequence complementary to the region between nucleotides 1401 and 1560 of the 18S rRNA. We hypothesize that Rta may assist 40S ribosome in recognizing the region I sequence to start a scanning process for Zta translation.