We previously immortalized primary human oral keratinocytes by transfection with cloned human papillomavirus type 16 (HPV-16) DNA and established a cell line, human oral keratinocytes-16B (HOK-16B). This line contained intact HPV-16 DNA in an integrated form, expressed viral genes, and demonstrated an indefinite life span. However, the cells proliferated only in keratinocyte growth medium containing a low level of calcium and were not tumorigenic in nude mice. To develop an in vitro multistage model suitable for the study of human oral carcinogenesis and to investigate the molecular mechanisms of cell transformation, the HOK-16B cells were exposed to either benzo(a)pyrene [B(a)P] or 7,12-dimethylbenz(a)anthracene (DMBA). Two chemically transformed cell colonies, one from B(a)P exposure and the other from DMBA treatment, were isolated. These cell lines proliferated well in Dulbecco's minimum essential medium containing a physiological level of calcium, demonstrated anchorage-independent growth, and developed tumors in nude mice. They contained integrated HPV-16 sequences and expressed higher levels of epidermal growth factor receptor (EGFR) and c-myc transcripts compared to the parental cells. Similar to their immortalized parental cells, the chemically transformed cells also contained lower levels of p53 protein and transforming growth factor-beta (TGF-beta) transcripts than normal human oral keratinocytes. These results indicate that malignant transformation of oral keratinocytes can be caused by a sequential combined effect of high-risk HPV and chemical carcinogens. It also demonstrates that overexpression of viral E6/E7, EGFR and c-myc messages, together with the down-regulation of TGF-beta mRNA and the inactivation of p53 gene, may be associated with the malignant conversion of HPV-immortalized cells.