The genome of the prototype stain of coxsackievirus B4 (J.V.B. Benschoten) has been cloned in Escherichia coli and its complete nucleotide sequence determined. Excluding the poly(A) tract, the RNA genome is 7395 nucleotides in length and appears to encode a single polyprotein of 2183 amino acids. The predicted amino acid sequence of the polyprotein shows close homology (88%) to that of the previously sequenced coxsackievirus B3 and to certain regions of the polyproteins of the polioviruses and human rhinovirus 14. This allows identification of putative polyprotein cleavage signals, antigenic domains and other structural features likely to be important to the biological integrity of the virus.