ABSTRACT High‐affinity Ca 2+ transport ATPases play a crucial role in controlling cytosolic Ca 2+ . The amyloid β‐peptide (Aβ) is a neurotoxic agent found in affected neurons in Alzheimer's disease (AD) that has been implicated in dysregulation of Ca 2+ homeostasis. Using kinetic assays, we have shown that the Ca 2+ dependencies of intracellular Ca 2+ ‐ATPase (SERCA and SPCA) activity are the same in human AD and normal brain but that of plasma membrane Ca 2+ ‐ATPase (PMCA) is different. The addition of Aβ to normal brain decreases the PMCA activity measured at pCa 5.5, resulting in the same Ca 2+ dependency as that seen in AD brain, whereas the addition of Aβ to AD brain has no effect on PMCA activity. Aβ also decreases the activity of PMCA purified from pig cerebrum, the effect being isoform specific. The level of inhibition of purified PMCA caused by Aβ is reduced by cholesterol, and the level of inhibition of PMCA activity by Aβ in the raft fraction of pig synaptosomal membranes is lower than for the nonraft fraction. We conclude that the effect of Aβ on PMCA activity could be important in amyloid toxicity, resulting in cytoplasmic Ca 2+ dysregulation and could explain the different Ca 2+ dependencies of PMCA activity observed in normal and AD brain.—Berrocal, M.,Marcos, D., Sepulveda, M.R., Perez, M., Avila, J., Mata, A.M. Altered Ca2+ dependence of synaptosomal plasma membrane Ca2+‐ATPase in human brain affected by Alzheimer's disease. FASEB J. 23, 1826–1834 (2009)