Cultures of vertebrate peripheral neurons have been used to address a variety of issues related to the cell biology of the neuron. They are particularly amenable to experimental manipulations, such as microinjection, and can be cultured under a variety of different conditions designed to meet the needs of the particular experiment. This chapter focuses on cultures of rat sympathetic neurons from the superior cervical ganglia and on cultures of chick sensory neurons from the lumbosacral dorsal root ganglia. Information is provided on methods for dissection, preparation of culture dishes and substrates, composition of media, relevant growth factors, reduction of nonneuronal contamination, and maintenance of the cultures.