OBJECTIVES: This study examined whether mediators from biopsies of human irritable bowel syndrome (IBS) colons alter intrinsic excitability of colonic nociceptive dorsal root ganglion (DRG) neurons by a protease activated receptor 2 (PAR2)-mediated mechanism. METHODS: Colonic mucosal biopsies from IBS patients with constipation (IBS-C) or diarrhea (IBS-D) and from healthy controls were incubated in medium, and supernatants were collected. Small-diameter mouse colonic DRG neurons were incubated in supernatants overnight and perforated patch current-clamp recordings obtained. Measurements of rheobase and action potential discharge at twice rheobase were compared between IBS and controls to assess differences in intrinsic excitability. RESULTS: Supernatants from IBS-D patients elicited a marked increase in neuronal excitability compared with controls. These changes were consistent among individual patients but the relative contribution of rheobase and action potential discharge varied. In contrast, no differences in neuronal excitability were seen with IBS-C patient supernatants. The increased excitability seen with IBS-D supernatant was not observed in PAR2 knockout mice. A cysteine protease inhibitor, which had no effect on the pronociceptive actions of a serine protease, inhibited the proexcitatory actions of IBS-D supernatant. CONCLUSIONS: Soluble mediators from colonic biopsies from IBS-D but not IBS-C patients sensitized colonic nociceptive DRG neurons, suggesting differences between these two groups. PAR2 signaling plays a role in this action and this protease signaling pathway could provide novel biomarkers and therapeutic targets for treatment.