Amyloid-β (Aβ) plays an important role in Alzheimer's disease (AD) progression and is associated with synaptic damage and neuronal death. Epidemiological and experimental studies indicate that hypercholesterolemia and hyperhomocysteinemia increase susceptibility to AD; however, the exact impact and mechanisms involved are largely unknown. Few studies have addressed the combined effects of the above compounds, which are considered to be risk factors for developing AD, on Aβ-induced neurotoxicity. The aim of the present work was to analyze the relationships between homocysteine (Hcy) and cholesterol and their role in Aβ toxicity in human neuroblastoma cells, as well as the mechanisms associated with this neurotoxicity. In addition to finding that Hcy is involved in cholesterol homeostasis in neurons, we demonstrate that the combined effect of cholesterol and Hcy in the presence of copper significantly increases the levels of reactive oxygen species and may render neurons more vulnerable to Aβ.