Coxsackievirus B3-induced myocarditis in different immunocompetent mouse strains was used as a model to investigate interrelationships between virus replication and development of chronic enteroviral heart disease. Using in situ hybridization to detect enteroviral RNA, we show that heart muscle infection is not only detected in acute myocarditis but is also detected during the chronic phase of the disease. Coxsackievirus B3 could evade immunological surveillance in a host-dependent fashion, thus inducing a persistent infection of the myocardium in association with ongoing inflammation. Patterns of acute and persistent myocardial infection were quantitatively assessed in one representative mouse strain (A.CA/SnJ, H-2f) by applying computer-assisted digital image processing; these patterns were then related to the extent of myocardial tissue damage as well as to inflammation. We observed a strong correlation, both spatial and temporal, between viral replication and development of myocardial lesions, indicating that acute and chronic myocardial injuries are a consequence of multifocal organ infection. Analysis of strand-specific in situ hybridization revealed that viral replication in persistent infection is restricted at the level of RNA synthesis. The described procedure for quantitating organ infection provides a powerful tool for evaluating virus-host interactions and will be of particular interest to those studying human enterovirus-induced cardiomyopathies.