Prion proteins (PrPc) are receptors for amyloid β 1-42 (Aβ1-42) oligomers, but we do not know the impact of Aβ1-42 binding to PrPc on the interaction of membrane-bound PrPc with molecules that regulate downstream biological pathways. Stability of the PrPc dimeric complex and subsequent intermolecular interactions with membranous or cytoplasmic molecules are important for physiological functions of PrPc including neuroprotection. The principal aim of this study was to determine whether homodimer lifetime of PrPc is affected by the presence of Aβ1-42 oligomers. Single-molecule imaging analysis was carried out by total internal reflection fluorescence microscopy in PrPc-transfected CHO-K1 cells in the absence or presence of characterized Aβ1-42 oligomers. The contribution of different Aβ1-42 oligomer conformations to Alzheimer's disease pathophysiology and to the associated neurotoxicity is unknown. To be precise, with the oligomeric species used in our study, we biochemically analyzed the molecular weight of oligomers formed from Aβ1-42 monomers under our experimental conditions. The lifetime of PrPc homodimers was 210 ms, and in the presence of Aβ1-42 oligomers, the lifetime was significantly reduced (to 92 ms). The reduction of PrPc homodimer lifetime by Aβ1-42 oligomers may impair PrPc-mediated downstream neuroprotective signaling.