Summary. -Homologs of Bombyx mori nuclear polyhedrosis virus (BmNPV) Bm67 gene ORF67 have been found in the genome of all lepidopteran nuclear polyhedrosis viruses, but their function is still not very clear. In order to analyze it we employed a bacmid harboring the complete BmNPV genome including the Bm67 gene and expressing infectious virus (wtBacmid) for the construction of its Bm67-deficient variant (Bm67-KO-Bacmid) using the Red recombination system and the Bm67-repaired variant (Bm67-Re-Bacmid) using the Bac-to-Bac system. By transfecting BmN cells with these bacmids we demonstrated that the Bm67-deficient virus did not generate infectious virus, while the repaired virus restored its infectivity, indicating that the Bm67 gene is essential for the formation of infectious budding virus (BV). Electron microscopy of BmN cells transfected with the abovementioned bacmids showed many mature rodshaped virus particles in both wtBacmid-and Bm67-Re-Bacmid-transfected cells but none in Bm67-KO-Bacmid-transfected ones. Moreover, the real-time RT-PCR showed that the deletion of Bm67 from wtBacmid significantly reduced the levels of viral genomic DNA and transcripts of viral early genes dnapol, ie-1 and lef-3 but not those of transcripts of late gene vp39 and very late gene p10. The finding that the Bm67-deficient virus generated reduced levels of infectious virus and transcripts of early dnapol gene but not those of late genes indicates that the Bm67 gene is essential for BmNPV replication.