The aim of this study was to examine the role of p16 in the pathogenesis of squamous carcinoma of the gynecologic tract. Squamous carcinoma and carcinoma in situ from the female genital tract were examined for the expression of p16 by paraffin immunohistochemistry. About 74% (40/54) of cases showed p16 expression. By primary site, 77% (23/30) of cervical, 67% (6/9) of vaginal and 85% (11/13) of vulvar primaries expressed p16, but two endometrial primary squamous carcinomas were negative (0/2). In addition, p16 was not identified in non-dysplastic tissue and low grade dysplasia. In cases where there were matched vaginal or vulvar and cervical primaries in a given patient, there was concordant positive p16 expression. It is concluded that p16 is frequently expressed in squamous carcinoma of the cervix, vagina and vulva, but not seen in cases of benign and low grade lesions. It may be a marker of transformation from a low to a high grade lesion. More cases of endometrial primaries need to be studied to see if these evolve by a p16-independent pathway.