The prototype Hill of echovirus 9, a human enterovirus, exhibits no pathogenicity for newborn mice in contrast to some other echovirus 9 strains isolated subsequently during epidemics. In this communication we report the first complete nucleotide sequence and construction of an infectious clone of echovirus 9. Aside from the 3' poly(A)-tract, the RNA genome is 7420 nucleotides (nt) in length and encodes a single polyprotein of 2193 amino acids (aa). The open reading frame extends from position 740 to position 7318 of the genome. Sequence comparisons to other enteroviruses reveal a strong overall amino acid identity to echovirus types 11 and 12 (in each case 76%). The proteolytic cleavage sites of the three major capsid proteins were determined after purification by HPLC and protein sequencing. A full-length clone coding for an infectious RNA transcript was constructed, and recombinant echovirus 9 particles could be isolated from the supernatant of transfected cell culture. It is shown that the recombinant virus, like the original prototype, is non-pathogenic for newborn mice and does not multiply in skeletal muscles.