The existence of a massive cholinergic projection from cells in the medical septal nucleus (MS) and nucleus of the diagonal band (DB) to the hippocampal formation has been recognized for some time. However, the actual percentages of cholinergic and non-cholinergic neurons in the MS and DB which project to the hippocampus have not been reported. A procedure which combines horseradish peroxidase (HRP) and acetylcholinesterase (AChE) histochemistry in the same tissue was used to determine these percentages in the rabbit. Less than 50% of the neurons in the MS and DB which were labeled with reaction product following an HRP injection into the dorsal hippocampus also stained for AChE. Moreover, 70% of all neurons containing HRP reaction product were located in the DB, but neurons in the DB could not be differentiated from those in the MS on the basis of size or morphology. These data are taken to indicate that much of the MS-DB hippocampal projection is not cholinergic. Substance P is suggested as another possible transmitter within this anatomical system.