We have transfected two strains of normal human epidermal keratinocytes, v and u, with a plasmid containing the HPV16 genome, and compared the growth and terminal differentiation capacity of the transfectants, vp and up, with the parental cells. vp and up contained integrated HPV16 DNA and expressed a number of viral mRNAs; unlike v and u, vp and up were aneuploid and had chromosomal alterations. Their growth rate was similar to the parental cells, although they reached a higher confluent density. In contrast to v and u, vp and up grew well in the absence of 3T3 feeders and after 14 months in culture have reached passage 60. vp and up contained desmosomes and keratin filaments, but their capacity for stratification was reduced, as were the proportions of cells expressing the terminal differentiation marker, involucrin. Involucrin mRNA levels in vp and up were lower than in the parental cells but increased after 24 h in suspension, as observed in the normal cells. u and v did not form colonies in soft agar, whereas up had a colony-forming efficiency of approximately 0.1% and vp of less than 0.1%. vp and up did not form tumours in nude mice; like the parental cells they formed cysts a week after injection, but the cysts were less well organized than those formed by v and u. In conclusion, we have obtained two lines of immortalized human keratinocytes that are not tumorigenic, but have a reduced capacity for terminal differentiation. The ability to compare matched parental and transfected cells offers considerable potential for further studies of the role of human papilloma viruses in carcinogenesis.