Cl−-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl− transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid β proteins (Aβs), on the Cl−-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid β proteins (1–40, 1–42 and 25–35) concentration-dependently (1–100 nm) and time-dependently (from 1 h to 6 day) decreased Cl−-ATPase activity and elevated intracellular Cl− concentrations ([Cl−]i), Aβ25–35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Aβ-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 µm wortmannin or 20 µm quercetin, but not by a PI 3-kinase inhibitor, 50 nm wortmannin or 10 µm LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Aβ-treated cells as compared with each control. In the Aβ-exposed culture, but not in control, stimulation by 10 µm glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Aβ-treatment on the neurotoxicity of glutamate. Thus, Aβs reduce neuronal Cl−-ATPase activity, resulting in an increase in [Cl−]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.