Aged (23-25-month-old) male Long-Evans rats were assessed for deficits in spatial cognition relative to young (4-6-month-old) rats. An in vitro autoradiography study was then conducted for muscarinic M2 sites using [3H]AF-DX 384 to assess binding in basal forebrain and brainstem areas where cholinergic neurons are localized. The analysis of basal forebrain included the medial septal/diagonal band region that provides cholinergic innervation of the hippocampus; the laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus were analyzed in the brainstem. A significant age-related reduction in M2 binding was found in both the basal forebrain and brainstem. Only the reduction in the basal forebrain, however, was correlated with spatial learning impairment. Although the basal forebrain and brainstem cholinergic systems are each vulnerable in normal aging, contributions to the behavioral effects of aging may be distinctive for the two systems.