Double-stranded cDNA was synthesized from hepatitis A virus (HAV) RNA and inserted into the Pst I site of pBR322. Restriction endonuclease digestion and cross-hybridization of fragments yielded a map of overlapping cloned cDNAs that included at least 99% of the viral genome. Molecular clones containing HAV cDNA were identified by hybridizing cloned cDNA to electrophoretically resolved RNA from uninfected and HAV-infected tissue culture cells. Cloned cDNA probes specifically hybridized to RNA from infected cells, and the predominant species identified had the characteristic genomic length of picornaviral RNA (approximately equal to 7,500 nucleotides). A partial sequence from the 3' end of the genome revealed 414 bases in an open reading frame followed by two closely spaced stop codons, a 60-base noncoding region, and a tract of poly(A).