Numerous studies have shown a lamina-specific termination of commissural fibers to the dentate gyrus in the inner molecular layer. However, the exact course and arborization pattern of individual fibers remained unknown. In this study, the commissural fiber tract to the dentate gyrus of the rat has been studied using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L), which labels individual axons and their collaterals. Following iontophoretic application of the tracer, anterogradely labeled fibers were followed through the posterior basal fornix and medial fimbria where they formed a dense fiber bundle. Labeled fibers then entered the dentate gyrus close to the medial blade of the granule cell layer where they separated and traversed the hilus. Only in those cases where the injection also involved CA3 pyramidal cells could axons arborizing in the hilus be observed. Typically, fibers that continued into the molecular layer did not arborize in the hilus. Upon their entrance into the molecular layer, these fibers changed direction, gave off several collaterals, and followed a new path parallel to the granule cell layer where they preferentially formed en passant contacts. These commissural fibers to the inner molecular layer terminated in a wide septotemporal (longitudinal) extension. However, a considerable number of fibers reached the outer molecular layer where some of them formed extensive arborizations. Moreover, these commissural fibers to the outer molecular layer appeared to be restricted to the hippocampal lamella, corresponding to the level of the contralateral injection site. These data suggest the existence of three commissural projections to the rat dentate gyrus: (1) commissural fibers to the hilus arising from CA3 neurons, (2) commissural fibers to the inner molecular layer, and, (3) commissural fibers to the outer molecular layer.