Amyloid beta protein (A beta), which accumulates in the senile plaques in the brain of Alzheimer's patients, is cytotoxic to neurons. A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in which a yellow redox dye, MTT, is reduced to purple formazan, is very sensitive to the effect of A beta. In primary hippocampal cultures, inhibition of MTT reduction starts within 2 h after the addition of low concentrations of A beta and reaches a plateau in 12 h. This effect of A beta is not blocked by Ca2+ channel blockers or in Ca(2+)-free medium. In contrast, lactate dehydrogenase (LDH) release and trypan blue exclusion, which are indices of cell death, start 3 days after exposure to high concentrations of A beta and are blocked by Ca2+ channel blockers such as Co2+, nicardipine, and diltiazem. When A beta was washed out from the medium after 12 h, MTT reduction recovers and LDH release does not occur, suggesting that a long-lasting inhibition of the cellular redox system may be required to induce cell death. These observations demonstrate that A beta toxicity consists of two phases-a Ca(2+)-independent early phase and a Ca(2+)-dependent late phase- and that the early phase may be required to induce the late phase.