The aim of this study was to validate the role of p16(INK4a) immunocytochemistry as a marker of cervical epithelial neoplasia in cytology and biopsy samples. During the period of 4 years a total of 501 immunocytochemistry stains in cytology and biopsy samples were performed. The cytology smears were obtained from two independent laboratories of women with low-grade squamous intraepithelial lesion interpretation of Pap test or ThinPrep. Additionally, a selection of paraffin blocks from cases diagnosed previously with squamous intraepithelial lesion and cervical cancer were immunostained. Among 312 cytology samples analyzed by immunocytochemistry, 36.2% were positive for p16(INK4a). Negative cases were followed-up with conventional Pap smear and 86.9% showed regression. Among positive patients, 14 underwent surgery and 100% exhibited squamous intraepithelial lesion. In additional immunohistochemical analysis of selected biopsies from our archive, we found that the ratio of positivity for p16(INK4a) increased from 40% in L-SIL/CIN1 to 69.8% in H-SIL/CIN2 to 100% in H-SIL/CIN3 and squamous cell carcinoma. A relatively low number (24%) of cases with squamous metaplasia revealed focal and/or sporadic staining in the superficial layers of the epithelium. All the cases of cervical adenocarcinoma and mesonephric carcinoma were positive for p16(INK4a). p16(INK4a) is a useful additional marker for interpretation of problematic Pap smears and reduces the variability during evaluation of suspicious biopsies of the uterine cervix.