Previously, controllable gene expression in baculovirus was not possible using an insect system. We found that this was due to a high background activation of minimal promoter by the viral polyhedrin upstream (pu) sequence. Here, by truncation of the pu sequence, regulatory gene expression was established through the tetracycline regulatory expression system. This novel system was used to test the stimulatory function of the polyhedrin promoter by the controlled expression of the late expression factor-2 (lef-2). To efficiently trace lef-2 expression and analyze suppression of this gene, the coding sequences of lef-2 and enhanced green fluorescent protein (egfp) were ligated together to generate a fusion protein, and an approximately 100-fold suppression of egfp-lef-2 expression was achieved by doxycycline treatment. A very low level expression of lef-2 was found to be sufficient for proper expression of polyhedrin promoter; however, progressively higher levels of lef-2 expression could stimulate much higher-than-original polyhedrin promoter expression in the viral genome. This system was found to exhibit significantly better suppression than the double-stranded RNA (dsRNA) strategy, and would be useful for expression of foreign or viral genes whose functions require the interaction of multiple and/or unknown baculovirus gene products.