Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid β peptide (Aβ). This peptide is derived from a large integral membrane protein, the β-amyloid precursor protein (βAPP), by proteolytic processing. The Aβ has previously been found only in the brains of patients with Alzheimer's disease or advanced aging. We describe here the finding that Aβ is produced continuously by normal processing in tissue culture cells. Aβ and closely related peptides were identified in the media of cells transfected with cDNAs coding for βAPP in a variety of cell lines and primary tissue cultured cells. The identity of these peptides was confirmed by epitope mapping and radiosequencing. Peptides of a molecular weight of ∼3 and ∼4 kDa are described. The 4 kDa range contains mostly the Aβ and two related peptides starting N-terminal to the beginning of Aβ. In the 3 kDa range, the majority of peptides start at the secretase site; in addition, two longer peptides were found starting at amino acid F(4) and E(11) of the Aβ sequence. To identify the processing pathways which lead to the secretion of these peptides, we used a variety of drugs known to interfere with certain cell biological pathways. We conclude that lysosomes may not play a predominant role in the formation of 3 and 4 kDa peptides. We show that an acidic environment is necessary to create the N-terminus of the Aβ and postulate that alternative secretory cleavage might result in the formation of the N-terminus of Aβ and related peptides. This cleavage takes place either in late Golgi, at the cell-surface or in early endosomes, but not in lysosomes. The N-terminus of most of the 3 kDa peptides is created by secretory cleavage on the cell surface or within late Golgi.