To establish a reactivation model of genital and central nervous system infection, 3- to 12-week-old outbred or BALB/c mice were inoculated vaginally with the HG-52 strain of herpes simplex virus type 2 (HSV-2). Primary infection was confirmed by serially positive vaginal cultures. Mortality in 6- and 12-week old infected mice was about 20%. In survivors, clearance of infectious virus was confirmed in serially negative vaginal cultures. At 6 weeks, immunosuppression of survivors with cyclophosphamide and antilymphocyte serum was begun. Recurrent virus shedding, monitored by daily vaginal cultures, was detected in the majority of animals. All mice became moribund or died, usually during the third to fifth weeks of immunosuppression. Brains and spinal cords from which all sensory ganglia had been removed were homogenized and inoculated onto cultures. One or both central nervous system (CNS) samples were virus-positive in nearly half of these mice, and cell-free virus was isolated from most positive brain and cord supernatants tested. Three-fourths of mice had evidence of virus reactivation with immunosuppression, as indicated by vaginal or CNS isolations, and by failure to isolate virus by identical means in matched infected, non-immunosuppressed controls. Vaginal, spinal cord and brain isolates occurred independently of one another in many immunosuppressed mice, and could not be predicted from presence or absence of external genital lesions during primary infection. These experiments show that with immunosuppression, reactivations of latent HSV-2 infections in mice can be detected in the genital tract and CNS, and provide a model to study productive, recurrent CNS infection and disease.