The medial septum (MS) is a structure best known for its role in theta rhythm generation in the �hippocampus. Theta rhythm is a relatively slow brain oscillation of 3–12 Hz that is important for memory formation and synaptic plasticity. Despite that the role of the MS in theta generation has been investigated for more than half a century, very little is known about how MS neurons contribute to this �phenomenon. In addition, exactly how hippocampal neurons participate in rhythmic activity in the MS remains relatively unexplored. Our poor understanding of theta generation is due in part to the challenging conditions offered by in vivo experimentation. To circumvent these obstacles, this chapter describes how to obtain a complete septo-hippocampal preparation in vitro to investigate theta generation. This method allows the study of theta rhythm with whole-cell and multiple field recordings in combination with precise pharmacology in vitro . We show that the preparation displays rhythmic oscillations in the hippocampus upon activation of the septum and, conversely, exhibits rhythmic activity in the septum following hippocampal stimulation. These results suggest that the septo-hippocampal preparation is a potentially powerful tool to investigate the cellular mechanism implicated in theta generation in vitro .