Synaptic dysfunction during early stages of Alzheimer's disease (AD) is triggered by soluble amyloid-beta (A beta) oligomers that interact with NMDA receptors (NMDARs). We previously showed that A beta induces synaptic protein loss through NMDARs, albeit through undefined mechanisms. Accordingly, we here examined the contribution of individual NMDAR subunits to synaptotoxicity and demonstrate that A beta exerts differential effects on the levels and distribution of GluN2A and GluN2B subunits of NMDAR in dendrites. Treatment of cultured hippocampal neurons with A beta(1-40) (10 mu M, 1 h) induced a significant increase of dendritic and synaptic GluN2B puncta densities with parallel decreases in the puncta densities of denritic and synaptic pTyr(1472)-GluN2B. Conversely, A beta significantly decreased dendritic and synaptic GluN2A and dendritic pTyr(1325)-GluN2A puncta densities and increased synaptic pTyr(1325)-GluN2A puncta densities. Unexpectedly, A beta treatment resulted in a significant reduction of GluN2B and pTyr(1472)-GluN2B protein levels but did not influence GluN2A and pTyr(1325)-GluN2A levels. These results show that A beta exerts complex and distinct regulatory effects on the trafficking and phosphorylation of GluN2A and GluN2B, as well as on their localization within synaptic and non-synaptic sites. Increased understanding of these early events in A beta-induced synaptic dysfunction is likely to be important for the development of timely preventive and therapeutic interventions.