The entry mechanism of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), a group II NPV, in cultured cells was examined. SeMNPV budded virus (BV) enters by endocytosis as do the BVs of the group I NPVs, Autographa californica (Ac) MNPV and Orgyia pseudotsugata (Op) MNPV. In group I NPVs, upon infection acidification of the endosome triggers fusion of the viral and endosomal membrane, which is mediated by the BV envelope glycoprotein GP64. However, the SeMNPV genome lacks a homolog of GP64 envelope fusion protein (EFP). A functional homolog of the OpMNPV GP64 EFP was identified in SeMNPV ORF8 (Se8; 76 kDa) and appeared to be the major BV envelope protein. Surprisingly, a 60-kDa cleavage product of this protein is present in the BV envelope. A furin-like proprotein convertase cleavage site (R-X-K/R-R) was identified immediately upstream of the N-terminus of the mature Se8 protein and this site was also conserved in the Lymantria dispar (Ld) MNPV homolog (Ld130) of Se8. Syncytium formation assays showed that Se8 and Ld130 alone were sufficient to mediate membrane fusion upon acidification of the medium. Furthermore, C-terminal GFP-fusion proteins of Se8 and Ld130 were primarily localized in the plasma membrane of insect cells. This is consistent with their fusogenic activity and supports the conclusion that the Se8 gene product is a functional homolog of the GP64 EFP.