In field crops in the southeastern United States, larvae of Heliothis virescens (F.) are often infected with ascoviruses, especially toward the end of the growing season. Ascoviruses are unusual in that they are difficult to transmit per os, and several studies have provided data indicating that these viruses are vectored mechanically by parasitic wasps during oviposition. In Georgia, three parasitoids commonly parasitize H. virescens larvae: Cardiochiles nigriceps Viereck, Campoletis sonorensis (Cameron), and Microplitis croceipes (Cresson). In the current study, we investigated the transmission of ascovirus by these parasitoids by using females that were collected in the field or reared in the laboratory. After a single oviposition by C. nigriceps in an H. virescens larva with a 4-d-old ascovirus infection, all subsequent healthy larvae parasitized by this female developed ascovirus infection. After oviposition in an infected larva, examination of C. nigriceps by using transmission electron microscopy showed that ascovirus virions and ascovirus vesicles adhered to the inner surface of the ovipositor. The ovipositor of M. croceipes was shorter than those of C. nigriceps or C. sonorensis, and this was correlated with a lower rate of ascovirus transmission by the former species. Observation of C. nigriceps populations in the field indicates this species survives even when ascovirus prevalence in H. virescens is high. Laboratory studies of this host–parasite–virus system showed C. nigriceps larvae survived infection of their host if parasitoid larvae were at least second instars at the time of infection. If an ascovirus infection in the first H. virescens host was no older 48 h, a C. nigriceps female sometimes did not transmit ascovirus to subsequent hosts. Exposure to environmental conditions of the field decreased the capacity of C. nigriceps to transmit ascovirus, and transmission also decreased over the longevity of female parasitoids.