Ascoviruses (AVs) belong to a family of double-stranded DNA viruses that infect Lepidoptera insects and cause the unique pathology of forming virion-containing vesicles in the hemolymph of infected hosts. Virions of AVs are large and contain more than 12 polypeptides. A gene, TnAV-CP, encoding the major structural protein of the Trichoplusia ni ascovirus 2a (TnAV-2a) was cloned by immunoscreening an expression library with antibodies against total TnAV virion proteins. TnAV-CP is an intronless gene with an open reading frame encoding a protein of 455 amino acids. Southern blot showed that it is a single copy gene. A 3.8 kb BamHI fragment containing the complete TnAV-CP gene was cloned and sequenced. Northern analysis detected the transcription of the 1.4 kb TnAV-CP mRNA from 24 h after infection. The predicted TnAV-CP protein was expressed in bacterial expression system and purified to homogeneity. The recombinant protein was used to affinity-purify specific antibodies from the antiserum. The purified antibodies reacted strongly with a single protein of approximately 52 kDa from the total TnAV virion proteins in a Western blot. This protein corresponds to the most abundant structural protein present in the virions of several AVs. Sequence comparison showed that TnAV-CP is most homologous to the putative capsid proteins from AVs infecting noctuid insects, less homologous to that of Diadromus pulchellus ascovirus 4a (DpAV-4a), further supporting the distinction of two subgroups within the family Ascoviridae. Phylogenetic analysis using the putative capsid protein suggested that AVs were closely related to members of Iridoviridae, which corroborated the result based on DNA polymerase delta sequences. The apparent differences between Ascoviridae and Iridoviridae in host range, virion morphology, and genome configuration, and the similarities in genes and methylation of genomic DNA were discussed.