Very late expression factor (VLF-1) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for high levels of expression of the very late genes p10 and polh, and evidence suggests VLF-1 may also be involved in viral DNA replication. In this study, investigations determined whether VLF-1 is essential for viral DNA replication by generating a vlf-1 knockout bacmid containing the AcMNPV genome through homologous recombination in Escherichia coli. Additionally, a vlf-1 repair bacmid was constructed by transposing the vlf-1 ORF and native promoter region into the polh locus of the vlf-1 knockout bacmid. After transfecting these virus constructs into Spodoptera frugiperda (Sf-9) cells, the vlf-1 knockout bacmid was unable to produce a viral infection while the repair bacmid propagated at wild-type levels. Experiments were performed to conclude whether the vlf-1 knockout phenotype was due to a defect in viral DNA synthesis or late gene transcription. Southern blot analyses determined that the vlf-1 knockout bacmid was able to replicate viral DNA but only to about one-third the level of wild-type or rescued controls. In addition, virion DNA was not detected in the supernatant of transfected cells, indicating that the DNA synthesized by the mutant virus was unable to assemble into virions that bud out of the cells. Analysis of viral gene transcription confirmed that late gene transcription was not affected by the vlf-1 knockout but transcription of the very late gene p10 was substantially reduced.