Mutations in genes encoding presenilin 1 and presenilin 2 account for the majority of cases of early-onset familial Alzheimer's disease. The presenilins have been localized to the endoplasmic reticulum and Golgi, but which of the multiple hydrophobic segments of the polypeptide chain span the lipid bilayer is unclear. To address this question, we have constructed a series of chimeric molecules in which a topologically neutral reporter protein (a C-terminal fragment of prolactin) containing three artificial glycosylation sites is fused to presenilin 1 following each of the 10 potential transmembrane domains identified in hydrophobicity plots. We have expressed these chimeras by translation in reticulocyte lysate containing canine pancreatic microsomes and by synthesis in transfected COS cells. Based on utilization of the glycosylation sites and sensitivity of the reporter to protease digestion, we provide evidence that presenilin 1 has six transmembrane segments with the N and C termini in the cytoplasm. This model provides important clues to the potential functions of different parts of the presenilin molecule and how these might relate to the pathogenesis of Alzheimer's disease.