Oestrogen is known to influence pyramidal cell spine synapse plasticity in the CA1 subfield of the hippocampus. Apart from direct oestrogen action on the hippocampus, oestrogen effects mediated by subcortical structures are known to be important. The purpose of this study was to investigate whether the medial septum diagonal band of Broca (MSDB) takes part in mediating oestrogen effects to the hippocampus. Special attention was given to the role of cholinergic MSDB neurons that project to the hippocampus, as a rather large population of them contains oestrogen receptors and, consequently, may be sensitive to oestrogen signals. Adult female rats were ovariectomized. Oestradiol- and cholesterol-filled cannulae (control) were implanted into the MSDB. To selectively eliminate the cholinergic population of MSDB neurons of oestrogen-treated animals, a group of rats was injected with 192 IgG-saporin (SAP) into the lateral ventricle 1 week before the cannula implant. Immunostaining with anti-choline acetyltransferase and parvalbumin (PA) showed that cholinergic but not PA-containing GABAergic neurons were substantially reduced in the MSDB of SAP rats. Comparative electron microscopic unbiased stereological analysis on the spine synapse density of CA1 area pyramidal cells was performed between all animal groups. Rats that received oestradiol-filled cannulae showed a higher (30%) spine synapse density than control animals. Oestrogen-treated rats that had received SAP treatment showed no significant difference to controls. Thus, this observation indicates that septo-hippocampal cholinergic neurons are involved in mediating oestrogen effects to the hippocampus. The relevance of this observation to mnemonic functions and Alzheimer's disease is discussed.