A transformed cell line (HE-IS) was established by transient supertransfection of a SV40-transformed human epidermal cell culture (HE-SL) (R. T. Su and Y.-C. Chang, 1989, Exp. Cell Res. 180, 117-133) with a recombinant plasmid containing an entire Harvey murine sarcoma virus DNA. HE-IS cells showed morphological alterations and less stringent growth requirements in a defined medium. A variant line (HE-TC), selected later from the HE-IS cells after a prolonged maintenance in serum-containing Dulbecco's minimal essential medium (DMEM), was found to be able to grow in either defined or serum-containing medium. Neither HE-IS nor HE-TC cells were able to form colonies in soft agar or induce tumors in athymic mice when inoculated subcutaneously. When grown in defined medium, all transformed cells (HE-SL, HE-IS, HE-TC) revealed a similar keratin pattern as analyzed by two-dimensional gel electrophoresis. However, significant reduction of keratins (notably K5) was detected in HE-TC cells maintained in serum-containing DMEM. The keratin pattern appeared to be regulated by the growth environment. Because of their nontumorigenic characteristics and defined growth properties, these transformed keratinocytes are likely to provide a suitable model system for examining the regulation of cytoskeleton protein synthesis.