Five foreskin-derived keratinocyte lines, immortalized by transfection of human papillomavirus (HPV16) DNA, were cytogenetically abnormal, exhibiting numerical deviations and altered chromosomes due to translocations, deletions, achromatic lesions, or partial duplications. Furthermore, all lines had cells with either homogeneously staining regions or double minute chromosomes, alterations associated with malignancy or drug resistance. None of these lines were tumorigenic in nude mice, showing that such alterations which are a manifestation of DNA amplification also occur in nonneoplastic cells. By in situ chromosome hybridization, viral sequences were identified on abnormal chromosomes at the junction of chromosome translocations, at achromatic lesions and within homogeneously staining regions and duplicated chromosome segments. Thus, for the first time in an experimental system, HPV16 integration into the cellular genome was associated with the induction of a subset of chromosome alterations. HPV16 integration that frequently occurred at fragile sites and near protooncogenes may be a critical alteration which confers a selective growth advantage and an indefinite proliferative potential to HPV-transfected cells.