In the shark, C-type natriuretic peptide (CNP) is the only cardiac natriuretic hormone identified and is a potent activator of Cl- secretion in the rectal gland, an epithelial organ of this species that contains cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. We have cloned an ancestral CNP receptor (NPR-B) from the shark rectal gland that has an overall amino acid identity to the human homologue of 67%. The shark sequence maintains six extracellular Cys present in other NPR-B but lacks a glycosylation site and a Glu residue previously considered important for CNP binding. When shark NPR-B and human CFTR were coexpressed in Xenopus oocytes, CNP increased the cGMP content of oocytes (EC50 12 nM) and activated CFTR Cl- channels (EC50 8 nM). Oocyte cGMP increased 36-fold (from 0.11 ± 0.03 to 4.03 ± 0.45 pmol/oocyte) and Cl- current increased 37-fold (from -34 ± 14 to -1,226 ± 151 nA) in the presence of 50 nM CNP. These findings identify the specific natriuretic peptide receptor responsible for Cl- secretion in the shark rectal gland and provide the first evidence for activation of CFTR Cl- channels by a cloned NPR-B receptor.