A monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was used to determine the morphological characteristics of cholinergic neurons and axon terminals within the rat septum. Light microscopy revealed numerous large fusiform or multipolar ChAT-immunoreactive neurons in the medial septal nucleus/diagonal band complex (MSDB). In contrast, virtually no immunostained cells were found in the lateral septum (Nc. septalis dorsalis and Nc. septalis lateralis). Fine immunostained fibers were most abundant close to the midline in the MSDB mainly following an ascending course. A few thin ChAT-immunoreactive fibers and terminallike pericellular punctate structures were observed in the inner part of the dorsal septal nucleus. Electron microscopy of ChAT-immunoreactive neurons revealed large cell bodies rich in cytoplasmic organelles. The cell nuclei regularly exhibited multiple invaginations of the nuclear membrane. Only rarely were terminals found that established synaptic contacts on the cell bodies of immunostained neurons. In contrast, numerous terminals formed synaptic contacts on immunoreactive dendrites. ChAT-immunopositive terminals were studied in thin sections from the MSDB and from the dorsal septal nucleus. In both regions they appeared as heavily immunostained vesicle-filled boutons that established symmetric and asymmetric synaptic contacts. In the dorsal septal nucleus immunostained terminals often showed a basketlike arrangement around immunonegative cell bodies. Our fine structural study provides evidence that cholinergic neurons in the MSDB are similar to cholinergic neurons in the basal nucleus and neostriatum, which have been described by other investigators. The presence of cholinergic synapses in the septal complex indicates that this region not only contains cholinergic projection neurons, but receives a cholinergic input itself.