Human papillomaviruses (HPVs) and cigarette smoking are epidemiologically associated with cervical cancer. We recently found that HEN-16 and HEN-16-2 HPV type 16-immortalized endocervical cells form tumors after treatment with cigarette smoke condensate and derived 2 tumor cell line cultures, HEN-16T and HEN-16-2T, respectively. Here, we examine the molecular pathologic effect of tumorigenesis. HEN-16T and HEN-16-2T exhibit unchanged status and expression of integrated HPV 16 DNA. However, the expression of the cytokeratin CK7 and CK13 endocervical cell markers is more homogeneous in monolayer and organotypic raft cultures after tumorigenesis. For the effect of retinoic acid on monolayers for growth inhibition, HEN-16T were significantly less sensitive than the normal and immortalized non-tumorigenic cells. HEN-16-2T were completely resistant. Moreover, the rafts from both tumorigenic cell line cultures were resistant to retinoic acid and continued to display thick rafts and homogeneous severe dysplasia/carcinoma in situ. In contrast, the non-malignant HEN-16 and HEN-16-2 rafts were thinner, and treatment with retinoic acid blocked the formation of severe dysplasia, reconstructing an epithelium resembling that of the normal endocervix. Our results support the significance of non-viral factors in the mechanism by which cigarette smoking induces tumorigenesis in the late stages of HPV-initiated progression to cervical cancer. Importantly, our data indicate that the sensitivity to retinoic acid of the HPV-containing endocervical cells is lost following tumorigenesis in vitro and possibly in women.