A DNA sequence upstream from the polyhedrin gene of baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) was found to strongly activate the expression of full or minimal promoters derived from AcMNPV and other sources. Promoters tested included the minimal CMV (CMVm) promoter from human cytomegalovirus, the full heat shock promoter from Drosophila, and the minimal p35 promoter from baculovirus. Deletion and mutagenesis analyses showed that this functional polyhedrin upstream (pu) activation sequence contains three open reading frames (ORFs) ORF4, ORF5 and lef2. By plasmid transfection, the pu sequence was able to confer high level luciferase expression driven by all of these full or minimal promoters in insect Sf cells. A known baculovirus enhancer, the homologous region (hr) of AcMNPV, further enhanced the expression of these promoters. Experiments showed that while multiple hr sequences function in an additive mode, pu and hr together function in a synergistic manner, which results in an extremely high level of 18,000 fold promoter activation. Furthermore, a modified CMVm promoter containing pu and/or hr was inserted into the baculovirus genome to drive the luciferase-coding region. The CMVm promoter expressed luciferase much earlier, and although expressed a little bit less protein than did the p10 promoter, the CMVm promoter gave rise to greater luciferase activity. Therefore, we have uncovered a cryptic viral sequence capable of activating a diverse group of promoters. Finally, these experiments demonstrate that synthetic sequences containing pu, hr, and different full or minimal promoters, can generate a set of essentially unlimited novel promoters for weak to very strong expression of foreign proteins using baculovirus.