This study investigates the relationship between the aggregation state of β-amyloid peptides (βAPs) and their neurotoxic properties in vitro. Synthetic βAPs were synthesized and their aggregation properties were assessed using light microscopy, electrophoresis, and ultracentrifugation assays. The results showed that few βAPs formed stable aggregates immediately after solubilization, but peptides containing the highly hydrophobic β29–35 region formed stable aggregates over time. In short-term neuronal cultures, toxicity was specifically associated with βAPs that exhibited significant aggregation. Additionally, the partial reversal of β1–42 aggregation resulted in a concomitant loss of toxicity. A synthetic peptide derived from islet amyloid polypeptide, which does not aggregate, exhibited no toxicity, suggesting that βAP-induced neurotoxicity is specific to aggregated βAPs. The correlation between βAP aggregation and neurotoxicity was observed in both short-term and long-term neuronal cultures but not in astrocyte cultures. These findings support the hypothesis that β-amyloid protein contributes to neurodegeneration in Alzheimer's disease.This study investigates the relationship between the aggregation state of β-amyloid peptides (βAPs) and their neurotoxic properties in vitro. Synthetic βAPs were synthesized and their aggregation properties were assessed using light microscopy, electrophoresis, and ultracentrifugation assays. The results showed that few βAPs formed stable aggregates immediately after solubilization, but peptides containing the highly hydrophobic β29–35 region formed stable aggregates over time. In short-term neuronal cultures, toxicity was specifically associated with βAPs that exhibited significant aggregation. Additionally, the partial reversal of β1–42 aggregation resulted in a concomitant loss of toxicity. A synthetic peptide derived from islet amyloid polypeptide, which does not aggregate, exhibited no toxicity, suggesting that βAP-induced neurotoxicity is specific to aggregated βAPs. The correlation between βAP aggregation and neurotoxicity was observed in both short-term and long-term neuronal cultures but not in astrocyte cultures. These findings support the hypothesis that β-amyloid protein contributes to neurodegeneration in Alzheimer's disease.