The ASCOVIRIDAE: is a family of large double-stranded (ds) DNA insect viruses that contains four species, the Spodoptera frugiperda (SfAV1), Trichoplusia ni (TnAV2), Heliothis virescens (HvAV3) and Diadromus pulchellus (DpAV4) ascoviruses. These are unique among insect viruses in that the primary means of transmission among their lepidopteran hosts is generally by being vectored mechanically by hymenopteran parasitoids. Ascoviruses are similar in virion structure, but their relationships with their parasitoid vectors vary from being opportunistic to obligate. Little is known, however, about the relatedness of these viruses to one another or to other large dsDNA viruses. We therefore cloned and sequenced the delta DNA polymerase gene of DpAV4, characterized it and compared it to 59 eukaryotic and viral delta and epsilon DNA polymerases. Phylogenetic analyses based on these genes revealed that the ascoviruses DpAV4 and SfAV1 formed a group of virus species distinct from, but closely related to, species of the family IRIDOVIRIDAE: Detailed analyses of the relatedness of ascovirus species based on conserved delta DNA polymerase motifs showed two groups within the family ASCOVIRIDAE:, one containing DpAV4 and the other containing SfAV1, TnAV2 and HvAV3, which was consistent with their host-vector relationships. Despite significant differences in capsid symmetry between ascoviruses and iridoviruses, these results suggest that these viruses may have originated from a common ancestral virus.