Baculovirus GP64 envelope glycoprotein is a specific major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. For promoter activity analysis in the baculovirus gp64 gene, two DNA fragments containing 437 and 439 bp upstream of 5' ends of the BmNPV and AcMNPV gp64 ORF were amplified by polymerase chain reaction and cloned, respectively. The sequence analysis indicated that two gp64 genes have both early (CAGT) and late (A/GTAAG) transcriptional start sites. By use of the plasmids with a reporter luciferase gene (Luc) driven by gp64 promoter to transfect insect cells, transient expression assay showed that pBmgp64Luc had high expression levels in permissive Bm-N cells and very low levels in non-permissive Sf-21 cells, while pAcgp64Luc had relatively high expression levels both in permissive Sf-21 cells and in non-permissive Bm-N cells. Furthermore, the transcription of both gp64 promoters appeared to be transactivated by 2.4-4 times in corresponding permissive cells by corresponding viral factors, separately. By inserting BmNPV homologous region-3 (hr3) into the downstream of luciferase reporter gene driven by gp64 promoter, it enhanced transcription from both gp64 promoters by 13 - 22 times in Bm-N cells and over 7000 - 14,000 times in Sf-21 cells, respectively. In the presence of BmNPV hr3, correspondingly, the viral factors transactivated the transcriptional activity from two promoters by about 73 - 78 times in corresponding permissive cells. It suggested that BmNPV hr3 plays an important role in co-activation with viral factors onto the gp64 promoter besides the functions of viral DNA origin and enhancer.