Mast cell infiltrates in tissues of vulvodynia are common, but they have not been characterized for criteria of neoplastic mast cell disease or correlated with patient's concomitant diseases associated with increased mast cells. Formalin-fixed specimens of 35 patients with vulvodynia were evaluated immunohistochemically with antibodies to CD 3,4,8,20,117c and human mast cell tryptase, and for WHO-criteria of neoplastic mastocytosis (>25% spindled mast cell, CD25 expression, point mutations of the c-kit gene (D816V), and chronically elevated serum tryptase levels). Only 20/35 specimens showed a T-lymphocyte dominant inflammatory infiltrate on HE-stained sections, but all showed mast cells. 4/35 biopsies showed <10 mast cells/mm2, 15/35 specimens 40–60 mast cells/mm2 and 16/35 specimens >60 mast cells/mm2 (average 80/mm2). Control tissue contained typically <10 mast cells/mm2. Spindling, CD25-expression, c-kit gene mutations, or increased serum tryptase levels were not detected. 26/35 (74%) patients had concomitant autoimmune diseases, psoriasis, atopy, various allergies, preceding infections. Independent of the subtype of vulvodynia, the majority of mast cell rich biopsies with >40 mast cells/mm2 were classified as a secondary mast cell disorder reflecting an activated immune system in 75% of vulvodynia patients. Patients with increased mast cells may benefit from medical therapy targeting mast cells.