Objectives: Immune activation may have an important pathogenic role in the irritable bowel syndrome (IBS). While little is known about immunologic function in functional dyspepsia (FD), we have observed an association between cytokine secretion by peripheral blood mononuclear cells (PBMCs) and symptoms in IBS. Upper gastrointestinal inflammatory diseases are characterized by enhanced small bowel homing α4-, Β7-integrin, chemokine receptor 9 (CCR9) positive T lymphocytes. We hypothesized that increased cytokine release and elevated circulating small bowel homing T cells are linked to the severity of symptoms in patients with FD. Thus, we aimed to (i) compare cytokine release in FD and healthy controls (HCs), (ii) quantify gut homing T cells in FD compared with HC and patients with IBS, and (iii) correlate the findings to symptom severity and gastric emptying. Methods: PBMC from 45 (Helicobacter pylori negative) patients with FD (Rome II) and 35 matched HC were isolated by density gradient centrifugation and cultured for 24 h. Cytokine production (tumor necrosis factor (TNF)-α, interleukin (IL)-1Β, IL-6, IL-10) was measured by enzyme-linked immunosorbent assay. CD4 α4Β7CCR9 T cells were quantified by flow cytometry in FD, HC and 23 patients with IBS. Gastric emptying was measured by scintigraphy. Symptom severity was assessed utilizing the standardized Gastrointestinal Symptom Score. Results: FD patients had significantly higher TNF-α (107.2±42.8 vs. 58.7±7.4 pg/ml), IL-1Β (204.8±71.5 vs. 80.2±17.4 pg/ml), and IL-10 (218±63.3 vs. 110.9±18.5 pg/ml) levels compared with HC, and enhanced gut homing lymphocytes compared with HC or IBS. Cytokine release and CD4α4Β7CCR9 lymphocytes were correlated with the symptom intensity of pain, cramps, nausea, and vomiting. Delayed gastric emptying was significantly associated (r0.78, P0.021) with CD4α4Β7CCR9 lymphocytes and IL-1Β, TNF-α, and IL-10 secretion. Conclusions: Cellular immune activation with increased small bowel homing T cells may be key factors in the clinical manifestations of H. pylori-negative FD.