The nucleotide sequence of the Heliothis virescens ascovirus (HvAV-3e) DNA genome was determined and characterized in this study. The circular genome consists of 186,262 bp, has a G+C content of 45.8 mol% and encodes 180 potential open reading frames (ORFs). Five unique homologous regions (hrs), 23 'baculovirus repeat ORFs' (bro) and genes encoding a caspase homologue and several enzymes involved in nucleotide replication and metabolism were found in the genome. Several ascovirus (AV)-, iridovirus- and baculovirus-homologous genes were identified. The genome is significantly larger than the recently sequenced genomes of Trichoplusia ni AV (TnAV-2c) and Spodoptera frugiperda AV (SfAV-1a). Gene-parity plots and overall similarity of ORFs indicate that HvAV-3e is related more closely to SfAV-1a than to TnAV-2c.