A new ascovirus was isolated from Spodoptera exigua in Indonesia and was tentatively assigned as a new species, Spodoptera exigua ascovirus 5a (SeAV-5a) according to the present ICTV ascovirus naming scheme based on DNA restriction fragment length polymorphism (RFLP), hybridization, formation of occlusion body, tissue tropism and host spectrum. SeAV-5a replicated primarily in the fat body of susceptible hosts. SeAV-5a could be transmitted to S. frugiperda, Pseudoplusia includens and Trichoplusia ni, but not to Heliothis virescens. Infection with SeAV-5a arrested growth of the hosts, but prolonged their survival, which continued up to 33 days. Clusters of virions were seen inside the characteristic vesicles. Occasionally, virions were contained within vacuoles (one to five per vacuole) and some virions were embedded in occlusion bodies. The size of the SeAV-5a virion was 347×134 nm; however, aberrant long secondary viral products were also seen. The presence of occlusion body and Southern hybridization and Western immunoblot analyses suggest that SeAV-5a is more closely related to S. frugiperda ascovirus 1a (SfAV-1a) than to Trichoplusia ni ascovirus 2 (TnAV-2). Certain regions of the 182 kb genome of SeAV-5a showed hybridization to that of SfAV-1a. Two fragments in each of the SfAV-1a EcoRI and HindIII digests hybridized to the SeAV-5a genomic DNA probe. Five to eight HindIII and EcoRI fragments in SeAV-5a DNA hybridized to the SfAV-1a genomic probe.