A quantitative evaluation of p16INK4A overexpression together with its topographical localization in the epithelium of cervical biopsies from non-neoplastic lesions and cervical intraepithelial neoplasias (CIN 1, 2, and 3) was obtained by the development of an objective and sensitive immunohistochemical assay with chemiluminescent detection (CL IHC assay). The cervical biopsy samples were also checked for the presence of human papillomavirus nucleic acids. The quantitative evaluation of p16INK4A expression was performed by combining 2 parameters: (1) intensity of the chemiluminescent-positive signal in the epithelium and (2) percentage of epithelium interested by the overexpression of p16INK4A, to obtain a p16INK4A expression score. A cut-off value was determined by using the receiver-operator characteristic analysis to distinguish between low-grade and high-grade CIN. Quantitative data showed that both p16INK4A expression parameters increased with worsening grades of CIN and, when combined to obtain the p16INK4A expression score, they showed a sharp discrimination among different lesions. The differences between the average p16INK4A score of CIN1 versus CIN2 (0.57 versus 1.05), of CIN1 versus CIN3 (0.57 versus 1.31) and of CIN1 versus CIN2/3 (0.57 versus 1.20) were statistically significant. The quantitative evaluation of p16INK4A expression by CL IHC assay could be therefore an interesting adjuvant method to distinguish different CIN grades and to predict the risk of progression of early CIN lesions.