Background & Aims: Corticotropin-releasing factor receptor-1 (CRF 1) mediates the stress-induced colonic motor activity. Less is known about the role of CRF2 in the colonic response to stress. Methods: We studied colonic contractile activity in rats and CRF2/, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute partial-restraint stress (PRS), and/or intraperitoneal injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF1 and CRF2 using immunohistochemical and immunoblot analyses. We measured phosphorylation of extracellular signal-regulated kinase 1/2 by CRF ligands in primary cultures of LMMP neurons (PC-LMMPn) and cyclic adenosine monophosphate (cAMP) production in human embryonic kidney-293 cells transfected with CRF1 and/or CRF2. Results: In rats, a selective agonist of CRF2 (urocortin 2) reduced CRF-induced defecation (>50%), colonic contractile activity, and Fos expression in the colonic LMMP. A selective antagonist of CRF2 (astressin 2-B) increased these responses. Urocortin 2 reduced PRS-induced colonic contractile activity in wild-type and CRF-overexpressing mice, whereas disruption of CRF2 increased PRS-induced colonic contractile activity and CRF-induced defecation. CRF2 colocalized with CRF1 and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of extracellular signal-regulated kinase in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF 1 (NBI35965) or astressin2-B, respectively. The half maximal effective concentration, EC50, for the CRF-induced cAMP response was 8.6 nmol/L in human embryonic kidney-293 cells that express only CRF1; this response was suppressed 10-fold in cells that express CRF1 and CRF2. Conclusions: In colon tissues of rodents, CRF2 activation inhibits CRF1 signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF 2 function impairs colonic coping responses to stress. © 2011 AGA Institute.