The possible intervention of nuclear proteins as cofactors of integrase-catalyzed integration of retroviral DNA into the host cell genome is not fully understood. Among various nuclear proteins, DNA topoisomerase II appears to be a plausible candidate. This hypothesis is supported by a series of evidence, including the fact that integration is markedly affected by the topology of the target DNA and mainly occurs in transcribed regions in which topoisomerase II is preferentially located. In an attempt to confirm the validity of this hypothesis, we have comparatively investigated the early stages of a recombinant Moloney murine leukemia virus (psi neo) in two related Chinese hamster cell lines (DC3F and R/DC3F) expressing different levels of both isoforms of topoisomerase II. R/DC3F is derived from the parental cell line DC3F and displays a resistant phenotype towards the usual anticancer topoisomerase II inhibitors (actinomycin D, doxorubicin, and taxol). Results show that the early stages of the retroviral cycle are markedly impaired in cells underexpressing topoisomerase II (R/DC3F). This alteration mimics Fv-1 restriction and is characterized by about a 6-fold decrease in viral DNA synthesis and total inhibition of viral genome integration. The specific impairment of integration in R/DC3F cells compared to DC3F cells is assessed by the absence of G418-resistant colonies upon viral infection and a lack of the viral genome in cellular nuclear DNA as detected by the PCR procedure. These features are observed in relevant infecting conditions leading, in both cell lines, to the same amount of linear viral DNA and to the occurrence of two long terminal repeats containing circular DNA in the nuclear fractions.