Plasmid pHBG-1 was generated by cloning BamHI-Aha III digested fragment of E. coli β-galactosidase into the BamHI site of plasmid pAcI. Wild type AcNPV DNA and supercoiled pHBG-1 were cotransfected into Spodoptera frugiperda, an insect cell line, and extracellular virus (ECV) was plague assayed. A plague purified recombinant AcNPV that did not generate polyhedra was able to form blue plaques in the presence of X-gal. This recombinant AcNPV carrying E. coli β-galactosidase gene would be useful to investigate the regulation mechanism of polyhedrin gene expression.