The pathogenic role of enterovirus in patients with idiopathic dilated cardiomyopathy has been determined through a molecular biologic approach. Sensitivity in the detection of viral genomes in tissues varied between conventional nucleic acid hybridization and polymerase chain gene amplification. To improve diagnosis, we developed a strategy for reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA. We synthesized two sequence-specific oligonucleotides, primer 1 (5'dACCGACGAATACCACTGTTA3') and primer 2 (5'dCCTCCGGCCCCTGAATGCGGCTAAT3'), complementary to the 5' conserved viral genomic fragments. Viral RNA was amplified by double PCR with these two primers and hybridized with a 32-P labeled inter-primer probe (5'dATGAAACCCACAGGCACAAAG3'). Using this strategy, we detected as little as 10(-8) micrograms of coxsackievirus B3 RNA after amplification with RT-PCR, but detected none in the plasma of eight healthy adults. Among 15 patients with idiopathic dilated cardiomyopathy, viral RNA could be detected in one out of 12 plasmas (8%) and three out of four explanted heart tissues (75%). In contrast, no viral RNA could be detected in six samples of myocardial tissue from patients with other heart diseases. The only patient who had viral RNA in his plasma also had viral RNA in his myocardium. Thus, the high incidence of viral RNA in these patients suggests a possible etiologic link between them. Correct selection of specific PCR primers and the application of double PCR can improve chances of diagnosing enteroviral infection.