Stable integration of polyoma viral DNA into the host-cell genome is a prerequisite for continuous expression of the transformed phenotype. In this study we have mapped the chromosomal location of integrated viral DNA sequences in a polyoma-induced mouse salivary gland adenocarcinoma. By in situ hybridization, a major integration site was assigned to chromosome 14, band B. The combined results from in situ hybridizations to metaphase chromosomes, primary tumors, and cultured tumor cells indicate the presence of both integrated and free polyoma viral DNA in the tumors. Cytogenetically, the tumors were characterized by a pronounced karyotypic instability. No abnormal cells with the same karyotype were observed in any of the tumors. Nevertheless, it was possible to recognize a preferential pattern of chromosomes variation with certain common recurrent and sporadic deviations. Clonal gains and/or losses of single chromosomes were seen in all tumors. It is concluded from these results that karyotypic instability may play an important role in the genesis and progression of polyoma virus-induced salivary gland tumors.