Nucleofection is a transfection method used to introduce substrates such as cDNA plasmids into primary cells or other cell lines. The method can be successfully applied to cells that are considered difficult to transfect or suffer from low transfection efficiency as seen with traditional transfection techniques. Neurons in primary cultures retain many properties of their in vivo state and therefore, in many instances, are considered better experimental systems than immortalized cell lines, thus becoming increasingly desirable cell types for biomedical research. However, being post-mitotic, primary neuronal cultures are particularly difficult to transfect using routine transfection reagents. There is therefore a growing need for the efficient delivery of expression vectors into such neuronal cultures. In this chapter we will discuss the application of nucleofection for the heterologous expression of genes in primary neuronal cultures. We also discuss the advantage of this technique relative to other conventional methods, and describe a reliable method for transfection of cultured rat dorsal root ganglion (DRG) and trigeminal (TG) neurons.