The RNA genome of Rhopalosiphum padi virus (RhPV), like other members of the Dicistroviridae, contains two open reading frames that are preceded by internal ribosome entry sites (IRESs). To compare the activities of the two RhPV IRESs in insect cells, a system was established for the in vivo transcription and translation of plasmid templates containing the IRESs. In this system, the two RhPV IRESs directed initiation of translation from bicistronic plasmids with equal efficiency. Competition was observed between the two IRESs when they were in cis in a bicistronic plasmid. A mutation that disrupted the 3'-proximal pseudoknot of the intergenic (IG) IRES reduced translation initiation in vivo. Similarly, mutations in the RhPV IG IRES disrupted its ability to bind 80S particles in vitro. The two IRESs preferentially labelled proteins of different masses in UV cross-linking experiments, illustrating the different translation initiation mechanisms employed by the two elements.