The p16INK4aa (CDKN2) tumor suppressor gene is altered in several tumor types, but the frequency and mechanism of inactivation are largely unknown for endometrial carcinomas. We therefore wanted to assess the pattern and prognostic impact of p16 protein expression and promoter region methylation in a population-based series of 316 endometrial carcinoma patients with long-term and complete follow-up. Nuclear staining of p16 protein was related to clinicopathological variables, tumor markers, patient survival, and the presence of promoter region methylation. Absent/minimal nuclear staining for p16 protein was found in 14% of the tumors. Methylation of the p16 promoter region was found in only one tumor (0.7%) in a subset of 138 cases studied. This tumor lacked nuclear p16 protein expression as well. Loss of nuclear p16 staining was significantly associated with increased age, high FIGO (International Federation of Gynecology and Obstetrics) stage, serous papillary or clear cell histological types, high histological grade, aneuploidy, low estradiol and progesterone receptor concentrations, high expression of Ki-67, high intratumor microvessel density, and strong nuclear p53 protein expression. The 5-year survival was 47% for patients with absent/minimal nuclear p16 expression (n = 39) compared with 81% for patients with moderate/high nuclear p16 expression (n = 247; P < 0.0001). In Cox proportional hazards regression analysis, nuclear p16 expression showed an independent prognostic impact in addition to FIGO stage, age, Ki-67 expression, and microvessel density, with an adjusted hazard ratio of 2.9 (95% confidence interval, 1.3-6.5). The other variables lost their prognostic impact when nuclear p16 staining was added to the Cox model. In conclusion, loss of nuclear p16 protein expression was associated with aggressive endometrial carcinomas and high proliferative activity (Ki-67) and was found to represent a strong and independent prognostic indicator. Methylation of the promoter region seems to be an uncommon mechanism of p16 inactivation in endometrial carcinoma.