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Vulvar vestibulitis, a subset of vulvodynia, is present in 15% of patients in a general gynecologic practice. Only a few studies have focused on pathologic features of vulvar vestibulitis and none have included a control group. Punch biopsies from the vulvar vestibule of 12 patients with an age range of 22 to 51 years (mean 28 years) and 12 age-matched controls were analyzed for histopathologic features and investigated for the role of probable etiologic factors including human papillomavirus (HPV). A chronic inflammatory infiltrate was present in all specimens from patients with vestibulitis, and was composed predominantly of T-lymphocytes with a small number of B cells and an admixture of plasma cells, mast cells, and occasional monocytes. T-helper suppressor ratio was normal. The infiltrate was mild in 5 patients, moderate in 1, and severe in 6. Minor vestibular glands were observed in 8 (66%) patients and were associated with a periglandular inflammatory infiltrate. Squamous metaplasia was observed in 4 (44%) patients. Epithelial hyperplasia was present in 10 (83%) patients with mild dysplasia in 2 (16%). Immunohistochemistry for immunoglobulins IgG, IgA, and IgM showed the presence of IgG-positive plasma cells in 75% of patients, suggesting chronic irritation, but an autoimmune etiology cannot be excluded or confirmed. Biopsies of control cases did not show any inflammatory infiltrate. In situ hybridization for HPV 6, 11, 16, and 18 was negative in the patient group as well as in the control group. We conclude that histopathologic abnormalities in patients with vulvar vestibulitis are the result of a chronic inflammatory reaction of the mucosa of the vulvar vestibule, for which the cause remains unclear.