Cattle persistently infected (PI) with noncytopathic (ncp) bovine virus diarrhea virus (BVDV) are at risk for developing fatal mucosal disease (MD), which is considered to occur after superinfection with antigenically homologous cytopathic (cp) BVDV. In this study, we intranasally inoculated four PI-animals, that were PI with 2 ncp BVDV strains with 105 TCID50 antigenically closely related cp BVDV. Two PI-animals were inoculated with 105 TCID50 ncp BVDV and one PI-animal with virus free cell culture medium. Two out of four PI-animals that were inoculated with cp BVDV, developed MD and were euthanized at day 17 and at day 24 after infection. Postmortem, both animals showed typical lesions of MD and cp BVDV was isolated. The other two PI-animals that were inoculated with cp BVDV did not develop MD and were euthanized at day 51. They showed ulcerations in the gastrointestinal tract, cp BVDV was isolated and neutralizing antibodies were detected. From the three PI-animals, that were inoculated with ncp BVDV or cell culture medium, cp BVDV was also isolated. Cross neutralization tests were performed and no antigenic differences could be detected between the cp strains isolated from the PI-animals. Lymphocyte subsets of these PI-animals were determined by flow cytometric analysis. Before superinfection, the percentages of γδ subsets were much higher in the PI-animals that did not develop MD than in nonviremic control animals and in the PI-animals that died of MD. From this study we conclude that the presence of antigenically closely related cp BVDV in PI-animals does not necessarily lead to the development of MD and that besides the antigenic relatedness between the persisting ncp BVDV and cp BVDV other factors, for instance the number of circulating γδ cells, might determine whether or not PI-animals develop MD.