Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for cervical cancer. Greater than 90% of these cancers originate in the cervical transformation zone (TZ), a narrow region of metaplastic squamous epithelium that develops at the squamocolumnar junction between the ectocervix and endocervix. It is unclear why the TZ has high susceptibility to malignant transformation and few studies have specifically examined cells from this region. We hypothesized that cells cultured from TZ are more susceptible to cellular immortalization, an alteration that contributes to malignant development. We cultured primary epithelial cells from each region of human cervix (ectocervix, endocervix and TZ) and measured susceptibility to immortalization after transfection with the complete HPV-16 genome or infection of HPV16 E6/E7 retroviruses. Cells cultured from each cervical region expressed keratin markers (keratin 14 and 18) that confirmed their region of origin. In contrast to our prediction, cells from TZ were equally susceptible to immortalization as cells from ectocervix or endocervix. Thus, increased susceptibility of the TZ to cervical carcinogenesis is not due to increased frequency of immortalization by HPV-16. We developed a series of HPV16-immortalized cell lines from ectocervix, endocervix and TZ that will enable comparisons of how these cells respond to factors that promote cervical carcinogenesis.