The aggregation of beta-amyloid (Aβ) into soluble oligomers is considered an early event in Alzheimer's disease. Furthermore, the presence of these aggregates seems to lead to neurodegeneration in the context of this disease. However, the mechanisms underlying Aβ-induced neurotoxicity are not completely understood. Primary cultures of pyramidal neurons have proven to be an excellent model system for the study of such mechanisms. These cultures provide a homogenous population of neurons that extend and differentiate axons and dendrites and that establish functional synapses among them. In addition, the neurotoxic effects of preaggregated Aβ can be easily analyzed both morphologically and biochemically. Here, we describe in detail the materials and methods used for the preparation and maintenance of primary cultures of hippocampal pyramidal neurons, as well as for the aggregation of and treatment with Aβ.