A new Kunitz-inhibitor, which is different from aprotinin was extracted from bovine lungs with methanol, further purified by affinity chromatography on trypsin-Sepharose CL-6B and by repeated cation exchange chromatography on CM-Sephadex C-25. The inhibitor, which is less basic than aprotinin was characterized by polyacrylamide gel electrophoresis and ion-exchange HPLC. The N-terminus is blocked by pyroglutamic acid (Glu-1). After enzymatic removal of this residue with pyroglutamate aminopeptidase, complete identity with the primary structure of aprotinin was established by sequencing the inhibitor, which had been oxidized with performic acid, and by sequencing a tryptic fragment. The occurrence of the inhibitor, which can be denoted as pyroglutamyl-aprotinin or Glu-1-aprotinin, but which cannot be distinguished from aprotinin regarding its inhibitory specificity, is obviously the result of a different proteolytic processing of the bovine aprotinin precursor. By using CD and NMR-techniques it was shown that the N-terminus of the inhibitor is blocked, and that the conformation and the internal mobility correspond with those of aprotinin.