Compartmented neuronal cultures allow experimenters to establish separate fluid environments for neuronal axons and the soma from which they emanate. Physical isolation of cell bodies and axons is achieved by culturing neurons in tri-chambered Teflon rings. Dissociated ganglia are plated in one end compartment of the trichamber, and axonal growth is guided underneath watertight silicone grease barriers into a separate compartment. Since the axons and cell bodies are located in different compartments, they can be infected and assayed separately. We describe the assembly and use of compartmented neuronal cultures for in vitro study of directional infection of neurons by alpha herpesviruses. Selective application of viral inoculum to only one compartment ensures that the remainder of the neuron is not contaminated by input inoculum. This allows for quantification of viral spread, and unambiguous interpretation of immunofluorescence and electron microscopy images.