Trichilemmoma is considered a benign neoplasm derived from the outer root sheath of the hair follicle. Although the histogenesis of the lesion is unknown, a relationship between human papillomavirus (HPV) infection and development of these tumors has been suggested on morphologic observations. In order to determine whether HPV is, in fact, present in these lesions, we have analyzed sections from 25 formalin-fixed, paraffin-embedded trichilemmomas for the presence of occult HPV DNA using ultrasensitive DNA amplification technology. Under carefully controlled conditions normally effective in detecting HPV from condyloma or verrucae, we were unable to detect an HPV-induced amplimer in agarose gels or their corresponding Southern blots using radiolabeled probes. Our results do not support the hypothesis that trichilemmomas arise from HPV infection.