The pathway from the entorhinal cortical region to the hippocampal formation has previously been shown to be comprised of two sub-systems, one of which projects predominantly to the ipsilateral fascia dentata and regio inferior of the hippocampus proper, and a second which projects bilaterally to regio superior. The goal of the present investigation was to determine if these two pathways might originate from different cell populations within the entorhinal area. The cells of origin of these entorhinal pathways were identified by retrograde labeling with horseradish peroxidase (HRP). Injections which labeled the entorhinal terminal fields in both the fascia dentata and regio superior resulted in the retrograde labeling of two populations of cells in the entorhinal area. Ipsilateral to the injection, HRP reaction product was found in the cells of layer II (predominantly stellate cells) and the cells of layer III (predominantly pyramidal cells). Contralateral to the injections, however, the reaction product was found almost exclusively in the cells of layer III. With selective injections of the entorhinal terminal field in regio superior, only the cells of layer III were labeled, but these were labeled bilaterally. Selective injection of the entorhinal terminal field in the fascia dentata, however, resulted in the labeling of cells of layer II, but not of layer III, and these cells of layer II were labeled almost exclusively ipsilaterally. A very small number of labeled cells in layer II were, however, found contralateral to the injection as well. No labeled cells were found either in the presubiculum or parasubiculum following injections of the hippocampal formation. These cell populations were found capable of retrograde transport of HRP, however, since cells in both presubiculum and parasubiculum were labeled following HRP injections into the contralateral entorhinal area. These results suggest that the projections to the fascia dentata originate from the cells of layer II, while the projections to regio superior originate from the cells of layer III of the entorhinal region proper. The very slight crossed projection from the entorhinal area to the contralateral area dentata probably originates from the small population of cells in layer II which are labeled following HRP injections in the contralateral area dentata.