A panel of neuronal cell lines was derived from tumors of the neonatal rat central nervous system (CNS) in 1974, and two of these lines are in wide use today. Both the B35 and B50 lines offer a number of advantages to researchers who study CNS neurons in culture: they are simple to grow, to differentiate, and to transfect. B50 cells have been used extensively in the study of neuronal cell death, toxicology, and differentiation, whereas B35 cells have proven useful in the molecular analysis of endocytosis and of signaling pathways, in particular those that guide axonal outgrowth and cell motility. This chapter provides protocols for growing and transfecting B35 cells, selecting stable transfectants, exploring protein function using an antisense approach, and assaying cell motility in a Transwell chamber. All of these protocols have been written for researchers who have some skill in basic cell culture techniques, but previous experience with cultured neurons is not required.