A significant member of early-onset familial type of Alzheimer's disease cases has been shown to be caused by dominant mutations in either of the two genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). These two proteins are highly homologous to each other and have been reported to be mainly localized to the membranes of intracellular compartments such as the endoplasmic reticulum. Information about the membrane topological structures of these proteins is indispensable for understanding their physiological and pathological roles. Although several models have been proposed previously, their precise membrane topologies remain unknown. In this study, we examined this issue in detail by expressing a series of C-terminally deleted PS1 mutants fused to the hydrophilic portion of Escherichia coli leader peptidase in vitro using a reticulocyte lysate in the presence of microsomal membranes. Our results predict that PS1 exists mainly in a seven membrane-spanning structure with its C-terminal end exposed to the luminal space. This was also confirmed by expressing these fusion proteins in cultured cells. We further showed that a ninth hydrophobic segment is tightly bound to the membrane without spanning it. Based on the above observations, we propose a novel "seven membrane-spanning and one membrane-embedded" topological model for presenilins.