We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2–3 h.