MHV-A59 causes a chronic demyelinating disease in mice which is accompanied by persistence of viral genome in white matter. As part of the investigation into the mechanism of viral persistence, infection of glial cells, probable targets for chronic infection, was studied by the use of mixed glial, enriched oligodendrocyte and enriched astrocyte cultures. Following MHV-A59 infection in vitro, approximately 10% of oligodendrocytes and 30% of astrocytes expressed viral antigens in the absence of overt cytopathic effect. All cultures released infectious virus for the lifetime of the cultures, for at least 45 days in the case of mixed glial cultures. Cultures derived from previously infected mice were similar to those infected in vitro with respect to percentage of cells expressing viral antigen and levels of infectious virus produced. These results show (1) that glial cells are early sites of infection in vivo as well as sites of infection in vitro cultures, and (2) that glial cells support a non-lytic but productive infection in vitro and thus may contribute to viral persistence in vivo.