Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to debilitating cognitive deficits. Recent evidence demonstrates that glutamate receptors are dysregulated by amyloid beta peptide (Aβ) oligomers, resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits. Although it is well accepted that neuronal death in AD is related to disturbed intracellular Ca(2+) (Ca(2+)(i)) homeostasis, little is known about the contribution of NMDARs containing GluN2A or GluN2B subunits on Aβ-induced Ca(2+)(i) rise and neuronal dysfunction. Thus, the main goal of this work was to evaluate the role of NMDAR subunits in dysregulation of Ca(2+)(i) homeostasis induced by Aβ 1-42 preparation containing both oligomers (in higher percentage) and monomers in rat cerebral cortical neurons. The involvement of NMDARs was evaluated by pharmacological inhibition with MK-801 or the selective GluN2A and GLUN2B subunit antagonists NVP-AAM077 and ifenprodil, respectively. We show that Aβ, like NMDA, increase Ca(2+)(i) levels mainly through activation of NMDARs containing GluN2B subunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca(2+)(i) rise induced by a high concentration of Aβ (1μM), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca(2+)(i) homeostasis. Moreover, Aβ modulate NMDA-induced responses and vice versa. Indeed, pre-exposure to Aβ (1μM) decrease NMDA-evoked Ca(2+)(I) rise and pre-exposure to NMDA decrease Aβ response. Interestingly, simultaneous addition of Aβ and NMDA potentiate Ca(2+)(I) levels, this effect being regulated by GluN2A and GluN2B subunits in opposite manners. This study contributes to the understanding of the molecular basis of early AD pathogenesis, by exploring the role of GluN2A and GluN2B subunits in the mechanism of Aβ toxicity in AD.