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