To clarify the pathogenic relationships between human papillomavirus (HPV) and vaginal intraepithelial neoplasia (VAIN), we examined 71 vaginal biopsy specimens by histopathology and immunohistochemistry and analyzed the presence of HPV DNA by blot hybridization at Tm 2 40ยฐC using an HPV 58 probe (PBM-58 method). We found 27 cases of VAIN in patients with previous hysterectomy or antecedent or concomitant cervical intraepithelial neoplasia (CIN) and 44 cases of VAIN in patients without any abnormal findings on the cervix and the vulva. Histopathologically, 53 of 71 cases were graded as VAIN I and 15 and 3 cases were VAIN II and III, respectively, while 59 cases showed positivity for HPV capsid antigen by immunohistochemistry. Using the PBM-58 method, all 71 VAIN cases harbored a single HPV type at more than 1,000 viral copies per cell. We identified 15 different types (HPV 16, 18, 30, 31, 35, 40, 42, 43, 51, 52, 53, 54, 56, 58 and 66). Furthermore, we molecularly cloned 7 novel prototypes (HPV 59, 61, 62, 64, 67, 69 and 71) from VAIN I. Our results are strongly indicative that HPVs are etiologic agents of VAIN, like in the case of CIN. The distinct manifestations of HPV infection in the vagina are discussed in comparison with those in the cervix.