To study the involvement of DNA topoisomerase (topo) II on nonhomologous (illegitimate) recombination, we examined the effect of topo II inhibitors on random integration of exogenous vectors into human chromosomes. We transfected human cell lines PA1, HeLa and EJ-1 with linearized plasmid pSV2neo by electroporation, treated with topo II inhibitors and determined the frequency of Geneticin-resistant (G418r) colonies. We found that three topo II inhibitors, etoposide (VP-16), ICRF-193 and amsacrine (m-AMSA), greatly enhanced the frequency of G418r colonies. These effects were maximally expressed by as little as 12 hrs treatment with the drugs. Similar enhancements were found with different vectors (closed-circular and linear), different cell types, or by different transfection methods (calcium precipitation and lipofection). In contrast, the inhibitor treatments did not affect the transient expression of chloramphenicol acetyltransferase and beta-galactosidase activity following transfection with pSV2CAT and pCH110, respectively. Southern blot analysis revealed that the integration pattern of transfected pSV2neo into PA1 chromosomes was random and not characteristic for each inhibitor. These results suggest that topo II inhibitors directly act at a nonhomologous recombination reaction, promoting the integration process of transfected vectors into human chromosomes. We discuss the enhancement mechanism with a special emphasis on DNA strand breaks induced by the inhibitors.