The aim of this study was to investigate the proteolytic processing of the amyloid precursor protein (APP) in polarized primary cultures of hippocampal neurons. We have used the Semliki Forest virus (SFV) vector to express human APP695 in hippocampal neurons, sympathetic ganglia, and glial cells. The latter two cells secrete little or no APP, whereas hippocampal neurons secrete two forms of APP695, which differ in sialic acid content and in their kinetic appearance in the culture medium. In addition, rat hippocampal neurons expressing human APP produced significant amounts of the 4 kDa peptide beta A4. After 3 hr of metabolic labeling, the relative amount of beta A4 peptide to total cellular APP was 5.3%. Fibroblasts expressing APP695 using the same SFV vector mainly produced a related 3 kDa p3 peptide, a nonamyloidogenic fragment. Remarkably, the hippocampal neurons also produced significant amounts of beta A4-containing C-terminal fragments (10-12 kDa) intracellularly. Radiosequencing showed that these fragments were created at a previously described beta-secretase cleavage site and at a cleavage site 12 residues from the N terminus of the beta A4 domain (Thr584 of APP695), which we named delta-cleavage. Based on the observation that mature hippocampal neurons produce two potentially amyloidogenic fragments and secrete substantial amounts of beta A4 when expressing human APP, our results strengthen the hypothesis that neurons play a central role in the process of beta A4 deposition in cases of Alzheimer's disease and in aged primates.