F proteins of group II nucleopolyhedroviruses (NPVs) are envelope fusion proteins essential for virus entry and egress. An F-null Helicoverpa armigera single nucleocapsid NPV (HearNPV) bacmid, HaBacDeltaF, was constructed. This bacmid could not produce infectious budded virus (BV) when transfected into HzAM1 cells, showing that F protein is essential for cell-to-cell transmission of BVs. When HaBacDeltaF was pseudotyped with the homologous F protein (HaBacDeltaF-HaF, positive control) or with the heterologous F protein from Spodoptera exigua multinucleocapsid NPV (SeMNPV) (HaBacDeltaF-SeF), infectious BVs were produced with similar kinetics. In the late phase of infection, the BV titre of HaBacDeltaF-SeF virus was about ten times lower than that of HaBacDeltaF-HaF virus. Both pseudotyped viruses were able to fuse HzAM1 cells in a similar fashion. The F proteins of both HearNPV and SeMNPV were completely cleaved into F(1) and F(2) in the BVs of vHaBacDeltaF-HaF and vHaBacDeltaF-SeF, respectively, but the cleavage of SeF in vHaBacDeltaF-SeF-infected HzAM1 cells was incomplete, explaining the lower BV titre of vHaBacDeltaF-SeF. Polyclonal antisera against HaF(1) and SeF(1) specifically neutralized the infection of vHaBacDeltaF-HaF and vHaBacDeltaF-SeF, respectively. HaF(1) antiserum showed some cross-neutralization with vHaBacDeltaF-SeF. These results demonstrate that group II NPV F proteins can be functionally replaced with a homologue of other group II NPVs, suggesting that the interaction of F with other viral or host proteins is not absolutely species-specific.