The thrombin receptor PAR-1 belongs to a unique family of G-protein coupled receptors that are activated by proteolytic cleavage. We studied the effect of PAR-1 activation in the regulation of ion transport in mouse colon in vitro. Expression of PAR-1 in mouse colon was assessed by RT-PCR and immunohistochemistry. To study the role of PAR-1 activation in chloride secretion, mouse colon was mounted in Ussing chambers. Changes in short circuit current (Isc) were measured in tissues exposed to either thrombin, saline, the PAR-1-activating peptide, TFLLR-NH₂, or the inactive reverse peptide, RLLFT-NH₂, prior to electrical field stimulation (EFS). Experiments were repeated in the presence of either a PAR-1 antagonist or in PAR-1-deficient mice in order to assess receptor specificity. In addition, studies were conducted in the presence of chloride-free buffer or the muscarinic antagonist, atropine, in order to assess chloride dependency and the role of cholinergic neurons in the PAR-1 induced effect. PAR-1 mRNA was expressed in full thickness specimens and mucosal scrapings of mouse colon. PAR-1 immunoreactivity was found on epithelial cells and on neurons in submucosal ganglia, where it was colocalized with both vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). After PAR-1 activation by thrombin or TFLLR-NH₂, secretory responses to EFS, but not those to forskolin or carbachol, were significantly reduced. The reduction in the response to EFS was not observed in the presence of the PAR-1 antagonist, in PAR-1-deficient mice, when chloride was excluded from the bathing medium, or when atropine was present. PAR-1 is expressed in submucosal ganglia in the mouse colon and its activation leads to a decrease in neurally evoked epithelial chloride secretion.