A human papillomavirus type 18 (HPV-18)-immortalized human keratinocyte cell line (1811) has been transformed to tumorigenicity in nude mice by treatment with the carcinogen nitrosomethylurea (NMU). The NMU transformants (1811-NMU-T) showed additional chromosome alterations as compared with parental 1811 cells, including 18q deletion in two of two 1811-NMU-T lines analysed. Restriction fragment length polymorphism (RFLP) analysis indicated that both 1811-NMU-T lines had lost one allele of the 18q deleted in colon cancer (DCC) tumor-suppressor gene. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that DCC expression was absent or barely detectable in the 1811-NMU-T cells as compared with 1811 or normal keratinocytes, suggesting that the remaining DCC allele in the 1811-NMU-T cells was also altered. These studies indicate that reduction or loss of DCC expression may be an important step in NMU transformation of HPV-immortalized cells to tumorigenicity.