Abstract We report here the discovery of two novel human platelet and megakaryocytic DAMI cell enzymes that have β-secretase-like activity. These activities could potentially effect cleavage of the amyloid precursor protein (APP) at the β-amyloid peptide N-terminus, by an EC 220.127.116.11-like metalloprotease, and the N terminus-1 position, by a serine protease. Thus both enzymes may generate the amyloidogenic β-peptide. Studies of intact and Triton X-100-lysed DAMI cells, as well as intact versus subcellular fractions of platelets, demonstrate the presence of these proteolytic activities. The resting platelet has (1) a surface serine protease, demonstrated by its ability to cleave a β-secretase substrate and by its inhibitor sensitivity; and (2) a metalloprotease, recognized by an antibody to EC 18.104.22.168, which resides intracellularly in the α-granule membrane, is translocated to the surface on activation, and shows β-secretase-like activity by cleaving the same substrate. This metalloprotease can also cleave recombinant APP to a potentially amyloidogenic fragment. Surface metalloprotease was identified in DAMI cells by flow cytometry and Western blotting with a specific anti-EC 22.214.171.124 monoclonal antibody, while activity was identified by using two β-secretase substrates. This article is the first to document two previously unknown endoproteinases with β-secretase-like activity in platelets and DAMI cells. These proteases are capable of effecting cleavage of APP and could therefore contribute to Aβ deposition in the cerebrovasculature.