Trypsins have been purified and characterized in a multitude of mammalian and nonmammalian species, but not in the domestic cat (Felis catus). In several species two or more isoforms of trypsin have been isolated. Feline trypsin was purified from feline pancrease by sulfuric acid extraction, ammonium sulfate fractionation, gel filtration and affinity chromatography on a benzamidine-activated sepharose gel. Feline trypsinogen was purified by sulfuric acid extraction, SDS-PAGE and electroelution. Only one isoform of feline trypsin and its zymogen could be demonstrated. Isoelectric focusing on agarose gel revealed an isoelectric point of greater than 10.0 for both feline trypsinogen and trypsin. The molecular weight of feline trysinogen was estimated at 22,600, while the molecular weight of feline trypsin was estimated at 21,000. The N-terminal amino acid sequence of feline trypsinogen was Phe-Pro-Ile-Asp-Asp-Asp-Asp-Lys-Ile-Val-Gly-Gly-Tyr-Thr-Asn-Arg. We conclude that cats either have only one isoform of trypsin or that other isoforms are present in minute quantities, undetectable by commonly used methods. We further conclude that feline trypsinogen and trypsin are cationic. Finally, the N-terminal amino acid sequence of the last 16 amino acid residues of feline trypsinogen is closely related to that of other mammalian species and the final 8 amino acid residues (termed trypsinogen activation peptide) are identical to those of canine cationic trypsinogen.