These experiments were directed at determining the proportion and distribution of cholinergic septal cells which project to the rat hippocampal formation. Injections of WGA-HRP were placed into different regions of the hippocampal formation and sections through the septal complex were processed for the simultaneous demonstration of the retrogradely transported marker and for choline acetyltransferase (ChAT) immunoreactivity. Preliminary analysis of adjacent normal series prepared either for the demonstration of ChAT or stained by the Nissl method demonstrated several distinct cell groups in the classically defined medial septal nucleus and vertical limb of the nucleus of the diagonal band. The groups of cells ranged from almost entirely ChAT-positive to entirely noncholinergic. On the basis of shape and size of the constituent cells, the ChAT-positive cells of the septal complex were divided into dorsal, intermediate, and ventral subdivisions. The proportion of retrogradely labeled cells that were also ChAT positive ranged from 22.8% to 77.4% in different experiments. When only the hippocampus and dentate gyrus are considered, this variation can largely be accounted for by the topographic organization of the septohippocampal projection. The medial, noncholinergic half of the medial septal nucleus projects primarily to the rostral or septal portions of the dentate gyrus and hippocampus, whereas the lateral half, in which the dorsal ChAT group is located, projects heavily to more temporal levels. Rostral portions of the hippocampus and dentate gyrus receive most of their cholinergic input from the ventral ChAT cell group which forms a major component of the vertical limb of the nucleus of the diagnoal band. While some ChAT-positive cells in the intermediate group project to the hippocampal formation, they are generally less numerous than those from the dorsal and ventral groups. However, in a control experiment in which the WGA-HRP injection was placed into the cingulate cortex overlying the rostral hippocampal formation, the intermediate ChAT group accounted for 71.2% of the double-labeled cells.