Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp(21)-Ile(22), yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp(21)-Val(22), yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.