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Invasive cancers of the vagina are relatively rare and often resistant to effective treatment. While studies on the more abundant premalignant lesions of the vagina and premalignant and malignant tumors of the vulva and cervix have shown a frequent association with human papillomavirus (HPV) DNA infection, lack of fresh tissue samples has precluded similar studies on malignant tumors of the vagina. Using mostly in situ hybridization, we have retroactively examined 14 formalin-fixed, paraffin-embedded biopsies of invasive squamous cell carcinomas of the vagina. We have found 21% of the samples to have HPV DNA. These findings confirm a role for HPV in malignancies of the entire female lower genital tract.