Prior studies of Ki-67, cyclin E, and p16 expression have suggested that these biomarkers may be preferentially expressed in cervical neoplasia. This study examined and compared the distribution of staining for these three antigens in 1) normal and reactive epithelial changes, 2) diagnostically challenging cases (atypical metaplasia and atypical atrophy), 3) squamous intraepithelial lesions (SIL), and 4) high-and low-risk human papilloma virus (HPV) type-specific SIL. One hundred four epithelial foci from 99 biopsies were studied, including low-grade squamous intraepithelial lesions (LSIL; 24), high-grade squamous intraepithelial lesions (HSIL; 36), mature or immature (metaplastic) squamous epithelium (29), and atrophic or metaplastic epithelium with atypia (15). Cases were scored positive for Ki-67 expression if expression extended above the basal one third of the epithelium, for cyclin E if moderate to strong staining was present, and for p16 if moderate to strong diffuse or focal staining was present. HPV status was scored by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of extracted DNA. Immunohistochemical findings were correlated with histologic and viral data. Overall, a histologic diagnosis of SIL correlated strongly with all of the biomarkers used (p <0.001). Positive scores for Ki-67, cyclin E, and p16 were seen in 68.4%, 96.7%, and 100% of LSILs and 94.7%, 91.6%, and 100% of HSILs, respectively. Positive predictive values of these three biomarkers for HPV were 82.4%, 89.5%, and 91.4%, respectively. The positive predictive value for HPV of either cyclin E or p16 was 88.7%. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Normal or reactive epithelial changes scored positive for the three biomarkers in 7.7%, 8.0%, and 12%, respectively. Limitations in specificity included minimal or no suprabasal staining for Ki-67 in immature condylomas and occasional suprabasal staining of reactive epithelial changes (10%), diffuse weak nuclear cyclin E staining in some normal or metaplastic epithelia, and diffuse weak basal p16 staining and occasional stronger focal positivity in normal epithelia. Ki-67, cyclin E, and p16 are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease. (Because cyclin E and p16 are most sensitive for LSIL and HSIL [including high-risk HPV], respectively, use of these biomarkers in combination for resolving diagnostic problems, with an appreciation of potential background staining, is recommended.)