Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from insect hosts have been previously identified. In order to gain insight into DNA replication of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV, Groups II), the essential cis-acting DNA segments were studied. A strain, named Se-4, was plaque-purified from SeMNPV isolate US1. PCR, ELT-PCR and REN showed that Se-4 was genetically relatively homogeneous and retained the full-length of a hypervariable region which is usually prone to deletion from SeMNPV genome. To study the stability of this isolate in vitro, Se-4 was serially passaged in the Se301 cell line up to 10 times without dilution. Intracellular viral DNA extracted from every passage was analyzed by REN. A novel 3.5 kb PstI fragment was observed in passage 7 and the relative intensities of the bands increased with subsequent passages. In passage 10, the molar ratio of the fragment was much higher than those of any other viral DNA fragments. This fragment was thus expected to contain an important cis-acting element for SeMNPV DNA replication. The fragment was cloned and sequenced and it was found that it overlapped 3 525 bp with the published SeMNPV genome sequence (GenBank AF169823), from 81 014 nt to 84 538 nt. The region contained the SeMNPV non-hr origin (ori) of DNA replication and some ORFs including partial vlf-1, partial p26, Se84 as well asSe83, Se85, Se86, which are unique to SeMNPV. As compared to Autographa californica MNPV (Groups I), which also generated hypermolar DNA fragments containing the non-hr ori by serial undiluted passage in IPLB-SF-21 cell culture, our results provided in vitro evidence that the non-hr ori may also play an important role in the viral infection cycle of Groups II NPVs.