β-amyloid peptide (Aβ) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Aβ readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Aβ aggregates or oligomers is still under investigation. In this article, we show that different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Aβ aggregates faster than Aβ prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Aβ aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Aβ fibrils formed with agitation. In addition, Aβ fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Aβ aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Aβ aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Aβ oligomers.