The approximately 4 kD (39-43 amino acid) polypeptide (amyloid beta protein, A beta) deposited as amyloid in Alzheimer's disease (AD) is derived from a set of 695-770 residue precursor proteins collectively referred to as the amyloid beta-protein precursor (beta APP). Using immunoblotting techniques, metabolic labeling, and sequencing we have analyzed beta APP derivatives in medium conditioned by: (1) human mononuclear leukemic (K562) cells expressing a model beta AP-bearing carboxyl-terminal beta APP derivative (2) human neuroblastoma (M17) cells transfected with constructs expressing full length beta APP and (3) M17 cells expressing only endogenous beta APP. In each case, we observed the release of a approximately 4 kD beta APP derivative essentially identical to the A beta found in AD amyloid. A similar, if not identical, beta APP fragment was readily detected in CSF from both Alzheimer's disease patients and controls. These observations indicate that the A beta is produced and released by normal processing of the beta APP. To determine if the production of A beta or A beta-tearing COOH-terminal beta APP derivatives is altered in cells expressing the mutant beta APPs linked to familial AD, we have compared M17 cells expressing wild type beta APP with those expressing mutant beta APPs (beta APP delta I or beta APP delta NL). After continuous metabolic labeling for 8 hours, cells expressing the beta APP delta NL mutant showed a 5-fold increase in the relative amount of an approximately 11.4 kD A beta-bearing carboxyl-terminal beta APP derivative, and they released 6-fold more 4 kD A beta into the medium. These observations provide strong evidence that: (1) the pathway producing A beta in cultured cells is highly relevant to AD and (2) the beta APP delta NL mutant causes AD because its processing is altered in a way that releases increased amounts of A beta.