Colorectal and small intestinal visceral hypersensitivity has been demonstrated in irritable bowel syndrome (IBS). Serine protease signalling via protease-activated receptor (PAR)-2 promotes hyperalgesia to mechanical distension. Furthermore, serotonergic pathways are involved in gastrointestinal visceral sensitivity. Abnormalities of serine protease and serotonergic signalling components have been identified in IBS colorectal mucosal biopsies. We determined the role of altered mucosal serine protease and serotonergic signalling in small intestine of IBS patients. Duodenal mucosal biopsies of 34 IBS patients (10 constipation-,11 diarrhoea-predominant and 13 alternating) and 20 healthy subjects (HS) were collected. Gene transcripts of PAR-2, trypsinogen IV, TPH-1, SERT (serotonin transport protein) and serotonin (5-HT(3)) subunits were quantified using real-time PCR and 5-HT content was measured by ELISA. Irritable bowel syndrome patients showed 1.5-fold higher trypsinogen IV mRNA level compared to HS (P = 0.016). SERT expression was 1.8-fold higher in IBS compared to HS (P = 0.007). Mucosal 5-HT content was 1.7-fold higher in IBS compared to HS (P = 0.015). The increase was 2.1-fold in IBS-C relative to HS (P = 0.018). Transcript levels of PAR-2, TPH-1 and 5-HT(3) receptor subunits did not differ between IBS and HS. In conclusion enhanced trypsinogen IV expression in IBS may cause increased PAR-2 activation. Increased SERT expression and mucosal 5-HT content in IBS suggest higher 5-HT availability. Both may contribute to small intestinal visceral hypersensitivity in IBS patients.