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In recent years, a new gluten- or wheat-related disease has emerged, a condition labeled "nonceliac gluten sensitivity" (NCGS) or "nonceliac wheat sensitivity" (NCWS). NCWS pathogenesis is still uncertain and attributed to very different mechanisms. We aimed to study the different T-lymphocyte subsets in the rectal mucosa of NCWS patients to demonstrate the possible contribution of adaptative immune response. Twelve patients (11 women, 1 man, age range 23-61 yr, median 32 yr) with a definitive diagnosis of NCWS were recruited at random for the present study. They underwent rectal endoscopy with multiple mucosal biopsies at the end of a double-blind placebo-controlled (DBPC) wheat challenge when they reported the reappearance of the symptoms. As controls we included 11 "healthy patients", sex- and age-matched with the patients who underwent colonoscopy evaluation for rectal bleeding due to hemorrhoids. Cells freshly obtained from rectal tissue were stained to detect anti-CD45, anti-CD3, anti-CD4, and anti-CD8. Furthermore, intracellular staining was performed with anti-tumor necrosis factor (TNF)-α, anti-interleukin (IL)-17, and anti-IL-22. Production of TNF-α by CD45 + , CD3 + , CD4 + , and CD8 + cells, as well as of IL-17 by CD4 + cells, was higher in the rectal tissue of NCWS patients than in controls. On the contrary, IL-22 production by CD8 + cells was lower in NCWS patients than in the controls. In NCWS patients diagnosed by DBPC wheat challenge, there is a complex immunological activation, with a significant role for the adaptive response. NEW & NOTEWORTHY Nonceliac wheat sensitivity (NCWS) is a syndrome characterized by symptoms triggered by gluten intake. The pathogenesis is still uncertain. Studies have shown a role for innate immunity. We demonstrated that production of TNF-α by CD45 + , CD3 + , CD4 + , and CD8 + cells and of IL-17 by CD4 + cells is higher in the rectal tissue of NCWS patients than in controls. We clearly demonstrated that in patients with NCWS there is a significant role for the adaptive response.