The association between human papillomavirus (HPV) type 16 and cervical squamous-cell carcinoma has been well documented, and HPV 16 DNA has been shown to immortalize human genital keratinocytes in vitro. Using a panel of cytokeratin(CK)-specific antibodies, we examined the CK expression pattern, an important characteristic of epithelia, of the stratified squamous epithelium reconstructed in vivo from monolayer cultures of 2 human ectocervical and 3 human foreskin keratinocyte cell lines immortalized by HPV 16 DNA. Whereas the abnormal epithelium formed from these grafts presented certain aspects of mature terminal differentiation, such as morphology and expression of CK10/11, the expression patterns for CK19, and especially CK13, were more obviously abnormal. CK18 expression was not detectable in any of the epithelia formed from the 5 cell lines. In contrast, for an HPV-16-immortalized endocervical cell line and the CaSki cervical-carcinoma cell line there was expression of CK18. Our results indicate that HPV-16-induced immortalization of keratinocytes is associated with disruption of the normal CK expression pattern in stratified squamous epithelium and that expression of particular CKs can be differentially disrupted.