Borna disease virus (BDV) is an unclassified agent that causes neurological disease in a wide range of animal species and possibly in humans. The infectious nature of BDV has been long established but, despite extensive progress on the pathogenesis of the infection, the aetiological agent is still uncharacterized. Recent studies have shown that BDV replicates productively in cultures of foetal rabbit glial cells (FRG) which produce a virus-specific protein that is easily detected immunocytochemically. This provides a marker for BDV infectivity. This cell culture system was used to investigate the replication cycle of BDV. The agent required at least 1 h to bind to and penetrate the cells and the antigen was detected 24 h later. Cycloheximide and actinomycin D inhibited production of the antigen in inoculated cells, indicating that both protein synthesis and a DNA-dependent function were required for the production of viral antigen. Cocultivation of BDV-infected FRG cells with Vero cells resulted in a persistent productive infection in the latter. Use of these cells showed that the infectious agent matured exclusively in the cytoplasm and within the plasma membrane of the cell. Antigen-laden nuclei did not have infectivity. These studies showed that BDV has the physical and replicative properties typical of conventional viruses but its mechanism of replication and site of morphogenesis may be unique.