The molecular cloning of double-stranded cDNA synthesized from the single-stranded RNA genome of the cardiotropic Coxsackie B3 virus (Nancy strain) is reported. Full-length reverse-transcribed cloned viral cDNA of approximately equal to 7500 nucleotides generated infectious antigenically identical Coxsackie B3 virus upon transfection of recombinant plasmid DNA into mammalian cells, demonstrating the molecular cloning of a biologically active viral cDNA copy. Furthermore, the cloned cDNA is characterized by restriction enzyme analysis and partial nucleotide sequencing of the 5' end. The Coxsackie B3 virus cDNA described can now be used to study the molecular basis of human enteroviral heart disease, and it provides a valuable diagnostic means for patients with suspected viral heart disease.