Twenty-nine human immunodeficiency virus (HIV)-infected patients with white, nonremovable lesions on the lateral border of the tongue, clinically suggestive of oral hairy leukoplakia (HL), were studied. In particular, the value of local antifungal therapy in establishing the diagnosis of HL was investigated. In 15 patients (52%) the lesions could be ultimately attributed to a candidal infection of the tongue. In 10 of the remaining 14 patients, a biopsy was obtained from lesions persisting after local antifungal treatment. In all biopsy specimens, the diagnosis of HL was confirmed by histopathologic examination and the demonstration of Epstein-Barr virus DNA by polymerase chain reaction, Southern blot hybridization, and DNA in situ hybridization. The present data confirm that the diagnosis of HL in HIV-infected patients cannot be reliably made on clinical criteria alone, but requires histopathologic confirmation including the demonstration of Epstein-Barr virus DNA, preferably by DNA in situ hybridization. However, with regard to the differential diagnosis of white, nonremovable lesions on the lateral border of the tongue in HIV-infected patients, the present study suggests that persistence of lesions after local antifungal therapy is highly suggestive of HL.