Two experiments were performed. In the first, the cholinotoxin, AF64A (0.5, 1.0 or 1.5 nmol/ventricle), or vehicle (3.0 microliters) was injected (ICV) bilaterally into male rats (n = 23). Choline acetyltransferase (ChAT) immunoreactive (IR) perikarya in the four subgroups of the septal complex were visualized by immunocytochemistry (PAP method) 28 days postinjection, and counted using a microprojector (x40). The 0.5 nmol/ventricle dose of AF64A significantly reduced (31%) the number of ChAT-IR cell bodies in the intermediate subgroup (rostral extension of the nucleus basalis/substantia innominata). Higher doses did not produce additional reductions. The highest dose (1.5 nmol/ventricle) of AF64A resulted in significant decreases in ChAT-IR cell bodies in the dorsal (51%) and midline (35%) subgroups (medial septum), but did not affect the number of ventral subgroup (diagonal band of Broca) ChAT-IR neurons. In the second experiment, electrolytic lesions were placed in the corpus callosum, cingulum and overlying cingulate gyrus, in order to simulate the nonselective damage seen following the 1.5 nmol/ventricle dose of AF64A. In comparison to the surgical controls (n = 3), the electrolytic lesions (n = 6) failed to significantly affect the number of ChAT-IR perikarya in any of the septal subdivisions. Thus the distinct subgroups of septal ChAT-IR neurons are differentially sensitive to the toxic effects of ICV administered AF64A: intermediate much greater than dorsal greater than midline much greater than ventral subgroup.