To systematically examine mucosal biopsies for differences in cytokine gene expression and protein secretion. The study included 59 females with irritable bowel syndrome (IBS) and 39, otherwise healthy, female volunteers presenting for colonoscopy. Colonic biopsies from subsets were studied by microarray analysis (IBS, n=9; controls, n=8), quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (IBS, n=22; controls, n=21), and ex vivo biopsy culture (IBS, n=28, controls, n=10). Biopsies from patients with active colitis were used as inflammatory disease controls. While gene array analysis revealed extensive overlapping between controls and IBS patients, reduced expression of genes linked to chemokine function was evident among the IBS patients alone. Differential expression was confirmed by qRT-PCR or ex vivo biopsy culture for 5 out of 6 selected genes. Reduced secretion of chemokines (IL-8, CXCL-9 and MCP-1) but not pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) was established on the basis of the ex vivo biopsy cultures. These findings were in marked contrast to the IBD patients who demonstrated increased production of both chemokines and pro-inflammatory cytokines. Despite the expected heterogeneity of the disorder, differences in mucosal chemokine signalling were evident in this cross-sectional study of IBS patients at the level of both gene expression and protein secretion, with IBS patients demonstrating a consistent deficit in the expression and secretion of chemokines known to play a critical role in mucosal defence.