Cultured hippocampal slices from rodents, in which the architecture and functional properties of the hippocampal network are largely preserved, have proved to be a powerful substrate for studying healthy and pathological neuronal mechanisms. Here, we delineate the membrane-interface method for maintaining organotypic slices in culture for several weeks. The protocol includes procedures for dissecting hippocampus from rat brain, and collecting slices using a vibratome. This method provides the experimenter with easy access to both the brain tissue and culture medium, which facilitates genetic and pharmacological manipulations and enables experiments that incorporate imaging and electrophysiology. The method is generally applicable to rats of different ages, and to different brain regions, and can be modified for culture of slices from other species including mice.