Human papillomaviruses (HPV) are known etiological agents of benign proliferation of the skin and mucosa (papillomas and warts). They have also been implicated in the development of cervical dysplasia and anogenital carcinoma. The close association of HPV type 16 DNA with the majority of cervical carcinomas suggests the involvement of the virus in this type of cancer. We have developed an in vitro multistep model for human epithelial cell carcinogenesis. Primary human epidermal keratinocytes acquired an indefinite lifespan in response to transfection with HPV 16 DNA but did not undergo malignant conversion. Addition of Kirsten murine sarcoma virus (Ki-MSV), which contains an activated K-ras oncogene, to these cells induced morphological alterations associiated with the acquisition of neoplastic properties. The frequency of transformation by Ki-MSV was markedly enhanced by the inclusion of glucocorticoid. At optimal conditions, a 125-fold stimulation was observed. These findings demonstrate the malignant conversion of human primary epithelial cells in culture by the cooperation of HPV type 16 and an activated ras oncogene and support a multistep process for neoplastic conversion. The availability of a human epithelial cell transformation model should facilitate studies of the interaction between HPV and human epithelial cells.