A major histopathological feature of Alzheimer's disease is deposits of a approximately 4-kDa beta-amyloid peptide derived by proteolytic processing from a precursor, the beta-amyloid precursor protein (beta-APP). Proteolytic cleavage of beta-APP within the approximately 4-kDa beta-amyloid domain permits the secretion of the amino-terminal portion of beta-APP while concomitantly producing a membrane bound approximately 9-kDa carboxyl-terminal fragment. We have characterized the proteolytic cleavage site for beta-APP secretion by amino acid sequence analysis of the approximately 9-kDa beta-APP carboxyl-terminal cleavage product produced by recombinant and native expression systems. Recombinant beta-APP was generated by a vaccinia virus expression system in CV-1 monkey fibroblasts; endogenous beta-APP was obtained using a fibroblast line derived from an individual with Down's syndrome. The sequences of both unlabeled and metabolically radiolabeled approximately 9-kDa fragment from CV-1 cells reveal that the major (60%) secretory cleavage site is after Lys16 of the beta-amyloid domain as reported previously; however, an additional cleavage site is seen after Phe19 (40%). Radiosequence analysis of the carboxyl-terminal fragment purified from Down's syndrome fibroblasts indicates cleavage sites after Phe19, Glu22, and Gly25 and not after Lys16. CV-1 cells expressing beta-APP mutants lacking 4 and 6 amino acids adjacent to Lys16 yielded approximately 9-kDa fragments with two identical cleavage sites, neither of which occurred after the retained Lys16 but were after Glu11 and His13. These data suggest that secretion of beta-APP involves multiple proteinases and that the composition of these proteinases may vary within different cell backgrounds.