vlf-1 is a baculovirus gene that regulates very late gene expression (J. R. McLachlin and L. K. Miller, J. Virol., 68, 7746-7756, 1994) and also plays a crucial role in the replication of the budded form of Autographa californica nuclear polyhedrosis virus (AcMNPV) (S. Yang and L. K. Miller, "Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene." Virology, 245, 99-109, 1998). To examine the influence of vlf-1 expression on baculovirus infection, we constructed recombinant viruses that expressed only low levels of VLF-1 and recombinants with vlf-1 under the control of different promoters. Viruses with mutant alleles of vlf-1 that produced low levels of VLF-1 replicated the budded form of the virus normally but produced no occlusion bodies. Thus, a higher concentration of VLF-1 was needed to activate very late gene expression than was needed to support budded virus production. By altering the level and/or timing of vlf-1 expression, the timing of polyhedrin gene (polh) expression, which normally occurs very late in infection, could be advanced or delayed. Early overexpression of vlf-1 increased the level of expression from the polh promoter but caused premature cellular disintegration. The data indicate that VLF-1 is the limiting factor in very late gene expression and that the level of VLF-1 controls the onset of occlusion.