A human keratinocyte cell line was established by transfecting neonatal foreskin keratinocytes of a Chinese with human papillomavirus (HPV) 16 DNA. As evidenced by the prolonged life span, the clone formation from a single cell, the piling up after prolonged culturing without passage and the chromosomal aneuploidy, this cell line possesses the biological characteristics of immortalization. The reason for obligatory growth requirements on epidermal growth factor (EGF) is not clear. The partial growth requirement on hydrocortisone for this immortalized cell line suggests that the glucocorticoid responding element of HPV 16 may play a role in cell immortalization. The constant over-expression of keratin 19 in this and other HPV 16 immortalized squamous epithelia indicates that it may serve as a useful marker for the potential malignant transformation of squamous epithelial cells. This immortalized cell line provides a model for investigating the factors and cofactors involved in carcinogenesis and differentiation of human epithelial cells.