We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34° C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca -resistant cells did not proliferate at the nonpermissive temperature (40° C), indicating that they depended on T antigen for their proliferation. The temperature-sensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40° C. The serum/Ca -resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca -resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.