The present study describes the efferent projections and terminal distributions within the retrohippocampal region of individual layers of the rat medial entorhinal area (MEA) as studied by the methods of anterograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L) and retrograde transport of the fluorescent dye Fast Blue (FB). Analysis of the PHA-L injections that were relatively well restricted to single layers of the MEA reveals very sparse projections to the parasubiculum, presubiculum, and subiculum, while numerous projections within the MEA are found. The course and the termination of the intra-entorhinal projections differ depending upon the particular layer under study, and marked differences are found between the deep and the superficial layers in terms of the divergence of their respective projections. However, the general intra-entorhinal orientation of these projections is essentially the same for all layers: longitudinal with a slightly oblique course, such that at ventral levels the center of a particular terminal field is always located lateral to the center of the respective PHA-L injection. PHA-L injections into layer II label axons running horizontally within this layer as well as within the deep part of layer I, and PHA-L injections into the medial sector of layer II reveal horizontal projections that innervate most of the second layer. The horizontal projections of layer III are more restricted than those of layer II but both layer II and III have prominent longitudinal projections directed ventrally. From layers II and III, numerous axons project to the deep layers (IV-VI) probably en route to extra-entorhinal structures, since no major terminal fields were detected in the deep layers. The PHA-L and the FB experiments show that the deep layers (in particular IV and VI) have far more divergent intra-entorhinal projections than have layers II and III. PHA-L injections into layers IV, V, and VI reveal widespread efferent projections to all of the more superficially located layers of the MEA in addition to projections to the lateral EA. The retrograde transport studies show that layers IV and VI are the major sources of these divergent projections and that cells situated throughout the entire medial to lateral width of these layers project to every sector of the retrohippocampal region. Taken together, the findings of the present experiments suggest that (1) all layers of the MEA have longitudinal projections directed primarily toward the ventral (or temporal) part of this cortex, (2) the projections of layers II and III are relatively restricted compared of the deeper layers.(ABSTRACT TRUNCATED AT 400 WORDS)