Ameloblastoma (AM) is recognized as a benign tumour but locally invasive with a high risk of recurrence. In vitro model systems for studying AM are limited due to the fact that AM cells grow poorly and begin to senesce early. Japanese researchers have reported the construction of an AM cell line, AM-1, by exposing cells to human papillomavirus 16 (HPV16) but retaining the potential of transformation. In this study, we used a retroviral infection method to over-express the human telomerase reverse transcriptase (hTERT) gene to acquire immortality of hTERT(+)-AM cells. Furthermore, it was revealed both by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot that the pathway of immortalization was loss of p16, not p53 or p21. Also, there was no evidence indicating that the hTERT(+)-AM cells underwent malignant transformation by the nude mouse tumorigenicity assay. Taken together, this hTERT-immortalized cell line may be a potentially valuable and reliable cell model for further study of the invasive properties of AM in vitro.