Secretin was purified from the first 50 cm of proximal intestine of a single dog. The purification sequence included extraction in acid-ethanol, differential precipitation with acetone, Sephadex gel filtration and three successive HPLC steps. Dog secretin has the following sequence: HSDGTFTSELSRLESARLQRLLQGLV. The underlined amino acid represents a substitution of Glu for Asp at position 15 from the NH2-terminus of pig secretin. Dog secretin is equal in potency to pig secretin in stimulating pancreatic ductal flow in a rat bioassay system.