The amino acid sequence of pancreatic trypsin from the spiny Pacific dogfish (Squalus acanthias) has been determined and compared with the sequences of bovine and porcine trypsin. Dogfish trypsin contains one less amino acid residue (222) than the other two enzymes. Two-thirds of the residues in corresponding positions in dogfish and bovine trypsin are identical and the sequences ofall three enzymes are homologous. Of the 223 amino acid residues of bovine trypsin, 77 are replaced without significant changes in function. Seven replacements, all conservative, occur in the interior of the protein; the remainder are on the surface. All residues known to be components of the active site of bovine trypsin are present in corresponding positions in dogfish trypsin. Comparison of the three enzymes suggests calcium binding sites in dogfish trypsin. A corrected sequence of bovine trypsin identifies residue 67 as Asn and residues 84-87 as Ser-Asn-Thr-Leu.