Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn631 to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACENQ) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACENQ was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 °C revealed that ACENQ was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn635-Ser636 bond, three residues N-terminal to the normal secretase cleavage site at Arg638-Ser639. These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.