BackgroundThe process of copulation in Lepidoptera is understudied and poorly understood from a functional perspective. The purpose of the present paper is to study the interaction of the male and female genitalia ofTortrix viridanaLinnaeus, 1758 via three-dimensional models of pairs fixed during copulation. Other techniques (confocal laser scanning microscopy, scanning electron microscopy and histology) were used to clarify the role of the organs involved in the process.ResultsThree-dimensional models based on micro-CT scanned copulating pairs were generated allowing visualisation of the position of the male and female counterparts, spatial changes during copulation, and the skeleto-muscular apparatus involved in the process. The male genitalia and their musculature are simplified in comparison with other lineages of the family, but the opposite is true for the female genitalia. The attachment of the couple is achieved only through flexion of the valvae, clasping the large and sclerotised sternite 7 of the female. The anal cone and socii of the male are in contact with certain parts of the anal papillae and the sterigma of the female. The long tubular vesica is inserted into the narrow posterior part of the ductus bursae. Its eversion is achieved by an increase in haemolymph pressure. A possible mechanism of stimulation of the female via pulsations of the diverticulum of the vesica was discovered. A compressed sclerotised area of the ductus bursae putatively serves as a valve controlling the transfer of ejaculated materials. Copulation progresses through two phases: in the first the vesica and its diverticulum are inflated by haemolymph, and in the second the diverticulum is not inflated, and the vesica is occupied by viscous ejaculated material. The formation of the multilayered spermatophore was observed, and we discovered that sperm is transferred very late in the copulation process.ConclusionsCopulation process in Lepidoptera is studied for the first time with three-dimensional reconstructions of couples ofTortrix viridana, used as a model species. The internal genitalia is the scenario of multiple interactions between male and female, but the external remain static. A possible mechanism of stimulation of the female internal copulation organs is proposed.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12983-023-00500-4.