The aim of this study was to assess the prognostic value of p16INK4a as a marker of post-conization relapse in patients treated for cervical intraepithelial neoplasia grade 3 (CIN 3). A retrospective study of 76 women with CIN 3 diagnoses, treated at the Hospital of Santa Casa de Misericórdia of São Paulo (Brazil) between January 2003 and September 2004, was performed. The study samples were obtained from cervical conization procedures, where paraffin blocks containing areas with the greatest amount of neoplastic tissue were selected. Immunohistochemical techniques were used on individual paraffin blocks for each case to detect p16INK4a protein expression. The p16INK4a cell counts were performed in 10 different high-amplification fields (400x) by light microscopy and total cell count expressed as number of cells per mm2. Patients involved in this study were followed up at the colposcopy outpatient unit for at least 48 months after cervical conization. The correlation of p16INK4a values with post-conization evolution in the patients (disease relapse or disease free) was determined. A significantly higher count of cells expressing p16INK4a was found in those patients with disease relapse during follow-up (p < 0.001). The variables age, number of gestations, and births correlated positively with number of cells expressing p16INK4a cells (p < 0.001; p = 0.001; 0.009, respectively). No correlation was found for the variables menopause, hormonal contraception, or smoking (p = 0.369, 0.425 and 0.853, respectively). p16INK4a can be considered a biomarker of cervical intraepithelial neoplasia grade 3 cases presenting high risk of relapse or evolution to invasive carcinoma.