p16 is a cyclin-dependent kinase-4 inhibitor that is expressed in a limited range of normal tissues and tumors. In recent years, immunohistochemistry with p16 antibodies has been used as a diagnostic aid in various scenarios in gynecologic pathology. Diffuse (as opposed to focal) positivity with p16 in the cervix can be regarded as a surrogate marker of the presence of high-risk human papillomavirus (HPV). In cervical squamous lesions, p16 is positive in most high-grade cervical intraepithelial neoplasia (CIN) and in some cases of low-grade CIN, usually those associated with high-risk HPV. p16 may be useful to identify small focal high-grade CIN lesions, to distinguish some cases of CIN involving immature metaplastic squamous epithelium from immature metaplastic squamous epithelium not involved by CIN and to distinguish high-grade CIN from benign mimics. Most cervical carcinomas of squamous, glandular, and small cell type are p16-positive. In cervical glandular lesions, p16 is useful, as part of a panel, in the distinction between adenocarcinoma in situ (diffusely positive) and benign mimics, including tuboendometrial metaplasia and endometriosis, which are usually p16-negative or focally positive. p16 may be used, in combination with other markers, to distinguish between a cervical adenocarcinoma (diffuse positivity) and an endometrioid-type endometrial adenocarcinoma (negative or focally positive). Some uterine serous carcinomas are diffusely positive. In the vulva, p16 is positive in HPV-associated vulval intraepithelial neoplasia (VIN) but negative in VIN not associated with HPV. Similarly, HPV-associated invasive squamous carcinomas are p16-positive, whereas the more common non-HPV-associated neoplasms are largely negative or focally positive. In the uterus, p16 positivity is more common and widespread in leiomyosarcomas than leiomyomas, and this may be a useful aid to diagnosis, although problematic uterine smooth muscle neoplasms have not been extensively studied. Metastatic cervical adenocarcinomas in the ovary are usually diffusely p16-positive, and because these may closely mimic a primary ovarian endometrioid or mucinous adenocarcinoma, this may be a valuable diagnostic aid, although p16 expression in primary ovarian adenocarcinomas of these morphologic subtypes has not been widely investigated. Some ovarian serous carcinomas, similar to their uterine counterparts, are p16-positive.