J. Neurochem.(2011)119, 1074–1085.AbstractAn increasing body of evidence suggests that endothelin‐converting enzyme‐2 (ECE‐2) is a non‐classical neuropeptide processing enzyme. Similar to other neuropeptide processing enzymes, ECE‐2 exhibits restricted neuroendocrine distribution, intracellular localization, and an acidic pH optimum. However, unlike classical neuropeptide processing enzymes, ECE‐2 exhibits a non‐classical cleavage site preference for aliphatic and aromatic residues. We previously reported that ECE‐2 cleaves a number of neuropeptides at non‐classical sitesin vitro; however its role in peptide processingin vivois poorly understood. Given the recognized roles of neuropeptides in pain and opiate responses, we hypothesized that ECE‐2 knockout (KO) mice might show altered pain and morphine responses compared with wild‐type mice. We find that ECE‐2 KO mice show decreased response to a single injection of morphine in hot‐plate and tail‐flick tests. ECE‐2 KO mice also show more rapid development of tolerance with prolonged morphine treatment and fewer signs of naloxone‐precipitated withdrawal. Peptidomic analyses revealed changes in the levels of a number of spinal cord peptides in ECE‐2 KO as compared to wild‐type mice. Taken together, our findings suggest a role for ECE‐2 in the non‐classical processing of spinal cord peptides and morphine responses; however, the precise mechanisms through which ECE‐2 influences morphine tolerance and withdrawal remain unclear.