Hairy leukoplakia, often seen in patients with acquired immunodeficiency syndrome, is strongly associated with Epstein-Barr virus (EBV) infection and questionably associated with human papillomavirus (HPV) infection. To date, most in situ hybridization (ISH) studies suggest that the EBV is localized only to the superficial squamous layers, favoring the theory of lingual infection by saliva rather than by reactivation of latent lingual infection. We describe 11 formalin-fixed, paraffin-embedded specimens from patients with lingual hairy leukoplakia that we examined for the presence of the EBV, HPV, cytomegalovirus, and human immunodeficiency virus. We used standard DNA ISH for the EBV and cytomegalovirus and polymerase chain reaction ISH for the EBV, HPV, and human immunodeficiency virus. The EBV was present in all 11 specimens according to polymerase chain reaction ISH studies but in only seven specimens according to conventional DNA ISH. Polymerase chain reaction ISH localized the EBV to the basal and parabasal layers in addition to the strong localization in the upper epithelial layers. No evidence for HPV or cytomegalovirus DNA was found. The human immunodeficiency virus was focally localized to rare superficial squamous cells in seven specimens. The presence of EBV DNA in basal and parabasal lingual cells, as well as localization of latency-associated proteins in these layers, suggests that hairy leukoplakia in patients with acquired immunodeficiency syndrome might represent a reactivation of latent lingual infection accompanied by a dramatic increase in viral copy number in the more mature, superficial, squamous cells. The human immunodeficiency virus was also found in seven specimens, but the significance of this new finding is uncertain and requires further study. There is no evidence to suggest that the HPV is involved in the development of hairy leukoplakia.