Virus was isolated from the lumen of the calyx region of ovaries in the parasitoid wasp Campoletis sonorensis (Hymenoptera: Ichneumonidae), and the nature of the viral DNA was analyzed. DNA purified from a homogeneous band of virus contained double-stranded superhelical molecules which were polydisperse in molecular weight. At least 25 different covalently closed circles were present, ranging in molecular weight from 4.0 x 106 to 13.6 x 106. The virus DNA was analyzed with restriction enzymes, and the nature of the genetic complexity was evaluated by Southern blot hybridization of native superhelical and relaxed circular virus DNA and of Salland HindIII-digested DNA. The data suggest that most of the variously sized covalently closed DNAs were composed primarily of nonhomologous sequences. The different size classes of covalently closed viral DNAs did not appear to exist in equimolar concentrations. However, there was no evidence from observation of virus particles in the electron microscope or from virus fractionation experiments that a mixture of viruses was present in the calyx fluid. The results from this study suggest, that the virus isolated from C. sonorensis, like those isolated from other endoparasitic hymenoptera, may belong to a new class of DNA viruses in which the genome is multipartite, with each DNA existing as a superhelical molecule. The virus-like particles which occur in many species of endoparasitic hymenoptera belonging to the superfamily Ichneumonoidea (for a review, see reference 34) (12, 15, 16, 20, 21, 28, 29, 31, 42) are currently classified as subgroup C baculoviruses (18). Virus replication in the female parasitoid is intranuclear and restricted to the nuclei of the calyx (a specialized region of the oviduct) epithelial cells (20, 21, 29, 31, 34). Virus is secreted from the calyx cells and accumulates to a high concentration within the calyx lumen as part of a "calyx fluid." It is injected with the parasitoid egg into host caterpillars during oviposition. Certain experimental evidence indicates that a component of the calyx fluid protects the parasitoid egg from encapsulation within the hemocoel of its habitual host (19, 29-34, 40, 41). A component(s) of the calyx fluid may also be involved in mediating some of the physiological changes that occur in the host after parasitization and which are apparently of some ultimate benefit to the parasitoid (5, 6, 9-11, 14, 38, 39). Vinson and co-workers initially demonstrated that the calyx fluid of Campoletis sonorensis prevents encapsulation of its eggs in its habitual