Patients undergoing radiotherapy for advanced cervical and endometrial cancer bear a considerable risk of developing vaginal preneoplastic lesions. Radiotherapy itself has been considered to have a role in the pathogenesis of vaginal dysplasia, although human papillomavirus (HPV) involvement has also been suggested. A series of 88 patients who underwent hysterectomy and were irradiated for gynecological cancer, including 43 with postradiation vaginal dysplasia at colposcopy and 45 without vaginal lesions, were included in this study. Detection and genotyping of HPV DNA in vaginal scraping were carried out by a PCR-based method and compared with colposcopic and cytological findings and with other clinical and laboratory data. Forty-two (97.7%) colposcopy-positive subjects and 6 (13.3%) colposcopically-negative patients were PCR-positive for high-risk HPV DNA (P < 0.000001). Twenty-two out of the 43 patients with colposcopic lesions showed an abnormal Papanicolau (PAP) test. Cytologic examination was negative in all colposcopically negative women. Type 16 HPV DNA was more frequent in patients with high-grade squamous intraepithelial lesions and in patients treated with external radiotherapy, whereas other types of high-risk HPV were more common in patients with low-grade lesions and in those treated with brachytherapy. When considering colposcopy as the standard for diagnosing vaginal dysplasia, HPV DNA testing was more sensitive than the PAP test. However, the specificity of the PAP test was higher with no false-positive case. In conclusion, vaginal preneoplastic changes in women post-hysterectomy and receiving radiotherapy for cervical, endometrial, and vaginal cancer represent an HPV-related nosologic entity. Whereas colposcopic examination can detect these preneoplastic lesions, HPV genotyping is a sensitive, inexpensive, and noninvasive method that may complement colposcopy and the PAP test. J. Med. Virol. 67:401–405, 2002. © 2002 Wiley-Liss, Inc.