Mast cells are protective against snake venom sarafotoxins that belong to the endothelin (ET) peptide family. The molecular mechanism underlying this recently recognized innate defense pathway is unknown, but secretory granule proteases have been invoked. To specifically disrupt a single protease function without affecting expression of other proteases, we have generated a mouse mutant selectively lacking mast cell carboxypeptidase A (Mc-cpa) activity. Using this mutant, we have now identified Mc-cpa as the essential protective mast cell enzyme. Mass spectrometry of peptide substrates after cleavage by normal or mutant mast cells showed that removal of a single amino acid, the C-terminal tryptophan, from ET and sarafotoxin by Mc-cpa is the principle molecular mechanism underlying this very rapid mast cell response. Mast cell proteases can also cleave ET and sarafotoxin internally, but such "nicking" is not protective because intramolecular disulfide bridges maintain peptide function. We conclude that mast cells attack ET and sarafotoxin exactly at the structure required for toxicity, and hence sarafotoxins could not "evade" Mc-cpa's substrate specificity without loss of toxicity.