Primary cultures of rat and mouse hippocampus and cerebral cortex are widely used to study neuronal properties such as axonal extension, synaptic transmission, and excitotoxicity. Short-term culturing of these neurons can be very straightforward and is perhaps easier than culturing cells lines once dissections are made and cell stocks are frozen. Long-term cultures of relatively pure neuronal populations require slightly more effort, but protocols are described that are less complicated than most published protocols. These include simpler ways to clean and coat coverslips, as well as using glia-conditioned medium to eliminate the need to make individual cocultures of neurons and glia. These methods consistently yield hippocampal and cortical cultures expressing dendritic spines and synapses that survive over 3 weeks in culture. For investigators employing biochemical assays where a fairly large amount of protein is necessary, cortical neurons may be especially attractive to use as large amounts of tissue are obtained and available for culture.