The presubiculum (PrS) plays critical roles in spatial information processing and memory consolidation and has also been implicated in temporal lobe epileptogenesis. Despite its involvement in these processes, a basic structure–function analysis of PrS cells remains far from complete. To this end, we performed whole-cell recording and biocytin labeling of PrS neurons in layer (L)II and LIII to examine their electrophysiological and morphological properties. We characterized the cell types based on electrophysiological criteria, correlated their gross morphology, and classified them into distinct categories using unsupervised hierarchical cluster analysis. We identified seven distinct cell types: regular-spiking (RS), irregular-spiking (IR), initially bursting (IB), stuttering (Stu), single-spiking (SS), fast-adapting (FA), and late-spiking (LS) cells, of which RS and IB cells were common to LII and LIII, LS cells were specific to LIII, and the remaining types were identified exclusively in LII. Recorded neurons were either pyramidal or nonpyramidal and, except for Stu cells, displayed spine-rich dendrites. The RS, IB, and IR cells appeared to be projection neurons based on extension of their axons into LIII of the medial entorhinal area (MEA) and/or angular bundle. We conclude that LII and LIII of PrS are distinct in their neuronal populations and together constitute a more diverse population of neurons than previously suggested. PrS neurons serve as major drivers of circuits in superficial (LII–III) entorhinal cortex (ERC) and couple neighboring structures through robust afferentation, thereby substantiating the PrS's critical role in the parahippocampal region. J. Comp. Neurol. 521: 3116–3132, 2013.