Most neurodegenerative disorders are associated with aggregation and accumulation of misfolded proteins. One of these proteins - tau, is involved in a number of pathologies including Alzheimer's disease and frontotemporal dementia. Aggregation and phosphorylation of tau have been shown to be a trigger for abnormal signal transduction and disruption of cellular homeostasis. Here, we have studied the effect of extracellular tau at different stages of aggregation in cortical co-cultures of neurons and astrocytes, to understand how this process affects tau pathogenicity. We found that the species formed after prolonged in vitro aggregation of tau (longer than one day) are able to stimulate ROS production through the activation of NADPH oxidase without decreasing the level of the endogenous antioxidant glutathione. The same late insoluble aggregates of tau induced calcium signals in neurons and a gradual increase in the ionic current of artificial membranes. Both tau-induced calcium signals and ROS production in NADPH oxidase were reduced in the presence of the inhibitor of voltage-gated calcium channels (VGCC) nifedipine. This suggests that insoluble aggregates of tau incorporate into the membrane and modify ionic currents, changing plasma membrane potential and activating VGCCs, which induces a calcium influx that triggers ROS production in NADPH oxidase. The combination of all these effects likely leads to toxicity, as only the same insoluble tau aggregates which demonstrated membrane-active properties produced neuronal cell death.