Phagocyte-derived S100 proteins are endogenous activators of innate immune responses. S100A12 binds to the receptor for advanced glycation end-products, while complexes of S100A8/S100A9 (myeloid-related proteins, MRP8/14; calprotectin) are ligands of toll-like receptor 4. These S100 proteins can be detected in stool. In the present study we analyse the release of S100A12 and MRP8/14 from intestinal tissue. Specimens from patients with Crohn's disease (CD; n = 30), ulcerative colitis (UC; n = 30), irritable bowel syndrome (IBS; n = 30) or without inflammation (n = 30) were obtained during endoscopy. After 24 h culture, S100A12 and MRP8/14 were analysed in supernatants. Endoscopic, histological, laboratory and clinical disease activity measures were documented. We found an increased spontaneous release of S100A12 from tissue in inflammatory bowel disease (IBD). The release of S100A12 into the supernatants was 28-fold enhanced in inflamed tissue when compared to non-inflamed tissue (mean 46.9 vs. 1.7 ng/ml, p < 0.0001). In active CD, release of S100A12 and MRP8/14 was strongly dependent on localization, with little release from sites of active ileal inflammation compared to colonic inflammation. This difference was more pronounced for S100A12 than for MRP8/14. S100A12 and MRP8/14 provoked up-regulation of adhesion molecules and chemokines on human intestinal microvascular endothelial cells (HIMECs) isolated from normal colonic tissue. The direct release of phagocyte-derived S100 proteins from inflamed tissues may reflect secretion from infiltrating neutrophils (S100A12) and also monocytes or epithelial cells (MRP8/14). Via activation of pattern recognition receptors, these proteins promote inflammation in intestinal tissue. The enhanced mucosal release can explain the correlation of fecal markers with disease activity in IBD.