Recombinant baculoviruses are suitable for the high-level production of large multi-protein complexes. A tri-cistronic expression vector was constructed by the inclusion of two internal ribosome entry sites (IRESs). In this novel polycistronic vector, one single polyhedrin promoter controlled the transcription of a tri-cistronic transcript. Also, the first cistron was translated through a cap-dependent mechanism, while the second and third cistrons were translated by the IRESs derived from the 5' UTR of Rhopalosiphum padi virus (RhPV) and Perina nuda virus (PnV), respectively. The ratio of tri-cistronic gene expression levels produced by the three translational initiation modules is about 2:1:1 (cap:PnV IRES:RhPV IRES). This study indicates that polycistronic genes can be co-expressed at the translational level as in prokaryotic expression system by baculovirus biotechnology.