On the basis of the detection of the enteroviral RNA in the hearts of patients with healed myocarditis and dilated cardiomyopathy, we investigated cardiac viral persistence in experimental myocarditis. Weanling C3H/He mice were given myocarditis by inoculation with coxsackievirus B3 (Nancy strain), and their hearts were examined by genomic studies and viral isolation up to the 180th day after inoculation. The virus was isolated from the heart until the 9th day. By slot-blot hybridization, viral RNA was also only detected until the 9th day in the heart. Specific DNA amplification using the polymerase chain reaction (PCR) was performed after a reverse transcriptase reaction, then followed by Southern blot hybridization with a 32P-labelled oligomer probe. This technique achieved the type-specific detection of coxsackievirus B3 even at a level of less than one of the 50% tissue culture infective dose (TCID50). With this technique, viral RNA was detected up to the 28th day after inoculation. Thus, the viral RNA persisted in the hearts of these mice even when infectious virus could no longer be detected.