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Calcitonin gene-related peptide (CGRP) mediates neurogenic inflammation and modulates intestinal motility. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein 1. We used RNA interference to elucidate the specific role of CLR in colonic motility and inflammation. Intramural injection of double-stranded RNA (dsRNA) against CLR (dsCLR) into the colonic wall at two sites caused the spatial and temporal downregulation of CLR in the colon within 1 day of dsRNA injection. Knockdown of CLR persisted for 7-9 days, and the effect of knockdown spread to ~2 cm proximal and distal to the injection sites, whereas control dsRNA injection did not affect CLR expression. Measurement of isometric contractions of isolated colonic muscle segments revealed that in control dsRNA-injected rats, CGRP abrogated contractions entirely and decreased resting muscular tone, whereas in dsCLR-injected rats, CGRP decreased muscle tone but slow-wave contractions of varying amplitude persisted. In trinitrobenzene sulfonic acid-induced colitis, rats with knockdown of CLR displayed a significantly greater degree of edema and necrosis than saline- or control dsRNA-injected rats. Levels of the proinflammatory cytokines TNF-[alpha] and IL-6 were markedly upregulated by trinitrobenzene sulfonic acid treatment. TNF-[alpha] mRNA levels were further increased in CLR knockdown rats, whereas levels of IL-6 were unaltered. Thus this study demonstrates that CLR is a functional receptor for CGRP. calcitonin gene-related peptide; RNA interference; trinitrobenzene sulfonic acid; cytokines; colon-specific; tumor necrosis factor-[alpha] doi:10.1152/ajpgi.00464.2006