Hepadnaviruses integrate in cellular DNA via an illegitimate recombination mechanism, and clonally propagated integrations are present in most hepatocellular carcinomas which arise in hepadnavirus carriers. Although integration is not specific for any viral or cellular sequence, highly preferred integration sites have been identified near the DR1 and DR2 sequences and in the cohesive overlap region of virion DNA. We have mapped a set of preferred topoisomerase I (Topo I) cleavage sites in the region of DR1 on plus-strand DNA and in the cohesive overlap near DR2 and have tested whether Topo I is capable of mediating illegitimate recombination of woodchuck hepatitis virus (WHV) DNA with cellular DNA by developing an in vitro assay for Topo I-mediated linking. Four in vitro-generated virus-cell hybrid molecules have been cloned, and sequence analysis demonstrated that Topo I can mediate both linkage of WHV DNA to 5'OH acceptor ends of heterologous DNA fragments and linkage of WHV DNA into internal sites of a linear double-stranded cellular DNA. The in vitro integrations occurred at preferred Topo I cleavage sites in WHV DNA adjacent to the DR1 and were nearly identical to a subset of integrations cloned from hepatocellular carcinomas. The end specificity and polarity of viral sequences in the integrations allows us to propose a prototype integration mechanism for both ends of a linearized hepadnavirus DNA molecule.