Cholinergic dysfunction is an early-onset feature of Alzheimer’s disease (AD), which has been suggested to cause, at least partly, the cognitive deficits observed. The accumulation of fibrillar β-Amyloid-peptides (Aβ) in senile plaques in the AD brain have been discussed to cause the specific degeneration of cholinergic cells. However, senile plaques are observed in later stages of AD, so a weak correlation between cholinergic degeneration and plaque formation exists. Recent studies provide evidence that also pre-aggregated forms of Aβ play a major role in mediating neurotoxicity. Using the neuroblastoma cell line SN56.B5.G4, the effect of different Aβ(1-42) aggregates on cell viability was investigated. The cell line expresses typical cholinergic characteristics and thus should represent an appropriate model for cholinergic neurons. Fresh Aβ(1-42), which contained predominantly low-molecular weight species, induced a concentration- and time-dependent toxicity in cholinergic cells. Aged Aβ(1-42), which was rich in fibrils showed no toxic effect, indicating that the neurotoxic effect of Aβ (1-42) depends on the aggregation stage of the peptide. It was shown that the low-molecular weight species induced apoptosis in cholinergic cells. Furthermore, fresh Aβ(1-42) generated oxidative stress by formation of reactive oxygen species in the cell line. To determine whether the neurotoxicity of fresh Aβ(1-42) differed from toxicity caused by oxidative stress on cholinergic neurons, a gene expression analyses using microarray technology was performed. The data evaluation showed that Aβ(1-42) and oxidative stress resulted in a different gene expression pattern. The toxicity mediated by Aβ(1-42) is probably not only induced by its oxidative potential. Many of the genes affected by Aβ(1-42) were involved in apoptosis- and stress regulating pathways (MAPK-, Bcl2-family members). The peptide also down regulated many genes present in the endoplasmatic reticulum (ER), Golgi apparatus and/or otherwise involved in protein modification and degradation, indicating a potential role of ER-mediated stress in Aβ-mediated toxicity. The mechanisms identified could presumably play a role in the cholinergic degeneration in AD. Moreover, a number of genes, which are known to be involved in AD were identified. Aβ(1-42) changed the expression of cholinergic genes (vesicular acetylcholine transporter), of the lipid- (apolipoprotein J) and zinc- (zinc transporter) metabolism. These findings demonstrate the applicability of this cell culture model in AD research. Most of the changes identified by microarray analysis were validated by quantitative real-time RT-PCR and the related proteins by Western-blotting/immunodetection. This study supports recent findings of a major role of low-molecular weight Aβ-species in mediating cholinergic neurodegeneration in AD. The low-molecular weight forms are neurotoxic, enhance apoptosis and (ER)stress in the cells, which may account in part for the early-onset cholinergic degeneration before abundant accumulation of plaque formation appears.