Positive allosteric modulation of α7-nAChRs is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca2+ signalling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator PNU120596 affects intracellular Ca2+ signalling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca2+ with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using MTT or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca2+ concentration; (iii) released Ca2+ from the ER by a Ca2+-induced Ca2+ release mechanism only in α7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca2+, overloading of intracellular Ca2+, and neuronal cell death.This article is protected by copyright. All rights reserved.