Low-cost, large-scale production of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) using continuous insect cell culture is seriously hindered by the accumulation of AcMNPV mutants. Specifically, few-polyhedra (FP) mutants, with a reduced yield of occluded virus (polyhedra) and decreased infectivity, usually accumulate upon passaging in cell culture. FP mutations result from transposon insertions in the baculovirus fp25k gene, leading to significantly reduced levels of FP25K protein synthesis. This study evaluated the effects of removing the transposon insertion sites from the wild-type baculovirus fp25k gene; the mutated virus was denoted Ac-FPm. Specifically, this study involved a detailed comparison of wild-type (WT) AcMNPV and Ac-FPm with regard to the proportion of cells having polyhedra, number of polyhedra per cell, the fraction of empty polyhedra, number of occlusion-derived viruses per polyhedron, number of nucleocapsids in the nuclei, FP25K protein synthesis and genetic analysis of the fp25k gene. Removal of TTAA transposon insertion sites from the fp25k gene stabilized FP25K protein synthesis and delayed the appearance of the FP phenotype from passage 5 to passage 10. Electron micrographs revealed that more virus particles were found inside the nuclei of cells infected with Ac-FPm than in the nuclei of cells infected with WT AcMNPV (at passage 10). Abnormalities, however, were observed in envelopment of nucleocapsids and virus particle occlusion within Ac-FPm polyhedra. Thus, the FP phenotype appeared in spite of continued FP25K protein synthesis, suggesting that mechanisms other than fp25k gene disruption can lead to the FP phenotype.