Although >60% of oral cancer cases are not related to human papillomavirus (HPV) infection, most studies of oral carcinogenesis in human cells in vitro are carried out with human oral keratinocytes immortalized by HPV DNA. To explore whether human oral keratinocytes can spontaneously transform without HPV infection, we attempted to establish spontaneously immortalized and tumorigenic-transformed human oral keratinocytes by serial subculture to the post-mitotic stage. Here we report two spontaneously transformed human oral keratinocyte lines from adult human gingival samples. These lines were obviously immortal (>140 passages) and transformed phenotypes in vitro. One of the lines, Spi-HOK1, remained non-tumorigenic in nude mice, whereas the other line, Spt-HOK80, showed tumorigenicity. These lines showed epithelial origi-nality, but did not contain high-risk types of HPV DNAs. On karyotyping, Spi-HOK1 was aneuploid with a unique stable marker chromosome. Both cell lines revealed a mutation in the p53 gene, loss of p21WAF1/Cip1 and overexpression of p-Rb-Ser807/811. These cell lines were resistant to cisplatin-induced apoptosis by suppressing induction of apoptotic proteins. These results clearly demonstrate that spontaneous immortalization and spontaneous tumorigenic transformation of primary human oral keratinocytes can occur in vitro without HPV infection and are associated with chromosomal alterations, p53 mutation and impaired apoptosis. To our knowledge, this is the first report demonstrating that the Spi-HOK1 and Spt-HOK80 lines are novel cell lines that are spontaneously transformed from primary human oral keratinocytes.