Several experiments compared the effects of excitotoxic lesions of the septal/vertical limb nuclei of the diagonal band of Broca (VDB) complex with those of the hippocampus (sparing the subiculum) on different forms of visual discrimination learning. The septal/VDB lesions, which produced significant reductions in choline acetyltransferase activity in the hippocampus and the cingulate cortex, impaired acquisition of a conditional visual discrimination in an operant chamber, while the hippocampal lesion had no effect, unless there was a delay interposed between the discriminative stimulus and the response. Neither lesion affected simple visual or spatial discrimination or reversal learning, also carried out in operant chambers, but both significantly impaired the acquisition and retention of a spatial navigation task (Morris water maze), with the septal/VDB lesions again producing greater deficits than the hippocampal lesions. Possible explanations for this surprising result are discussed and it is concluded that; (1) additional cholinergic de-afferentation of the cingulate cortex produced by the septal/VDB lesion is of functional significance; (2) this may lead to deficits in conditional rule learning, which can contribute to spatial navigation performance under certain circumstances; and (3) the contribution of septal-hippocampal cholinergic projections to spatial learning is in need of re-appraisal.