The objective of the study was to determine whether vestibular fibroblasts from vulvar vestibulitis (VVS) patients produce higher proinflammatory cytokine levels when provoked with Candida albicans (yeast) and alpha-melanocyte-stimulating hormone (α-MSH) in vitro.Twenty anatomically defined fibroblast strains from patients and age-matched controls were stimulated with 5 regimens: no stimulus, α-MSH, heat-killed yeast, α-MSH plus yeast, and interleukin (IL)-1β. Supernatant products included the following: granulocyte macrophage colony-stimulating factor, interferon-γ, IL-10, IL-12, IL-1β, IL-2, IL-4, IL-6, IL-8, and tumor necrosis factor-α were assayed.Coincubation with α-MSH plus yeast significantly increased IL-6 (3-fold) and IL-8 (greater than 40-fold) production in patients and controls. Vestibular fibroblast exceeded external vulvar fibroblast production of IL-1β, IL-6, and IL-8 following yeast alone and α-MSH plus yeast stimuli in patients and controls. Substratified by anatomic origin, vestibular fibroblasts from VVS patients produced the highest relative levels of IL-1β, IL-6, and IL-8 at baseline and following the yeast-alone regimen.Localized pain of VVS may results from regionally elevated cytokines produced by vulvar vestibule–specific fibroblasts.