Organotypic slice cultures of the brain are widely used as a tool to study fundamental questions in neuroscience. In this chapter, we focus on a protocol based on organotypic slice cultures of mouse entorhinal cortex and hippocampus that can be employed to study axonal regeneration and collateral sprouting in the central nervous system in vitro. Using pharmacological as well as genetic approaches, axonal regeneration and sprouting can be influenced, and some of the molecular and cellular mechanisms involved in these processes can be identified. The protocol describes in detail (1) the generation of organotypic entorhino-hippocampal slice cultures, (2) the conditions needed for the analysis of axonal regeneration and collateral sprouting, respectively, (3) the lesioning technique, (4) tracing techniques to visualize regenerating entorhinal axons, and (5) an immunohistochemical technique to visualize sprouting fibers.