Infection of fibroblast cell lines initiated from BALB/c or NFR mice with coxsackievirus B3 (CBV-3) or B4 (CBV-4) resulted in infections which persisted for a limited number of subpassages of the infected cells in most cases, but for over a year in one case. In all instances primary acute infections were characterized by cytopathology and release of infectious virus progeny. Viral antigen could be detected during the acute phase of infection, but not in subcultured infected cells. Infectious center assays showed that every cell was infected during the acute phase of infection, but that from the first subcultivation on, the numbers of cells which were able to initiate infection were greatly reduced. The long term persistent CBV-3 infection was characterized by wide fluctuations in titers of virus released into the supernatant fluids. Interferon did not appear to play a role in maintenance of the persistent infection. Information derived from studies on mechanisms of CBV persistence in the in vitro model may help to elucidate the role of CBV in chronic human diseases such as myocarditis.