Microdissection techniques were utilized to measure the activity of choline acetyltransferase (ChAT) (enzyme responsible for synthesis of acetylcholine) in individual basal forebrain nuclei of aged (24 month) and young (4 month) male and female rats. Small but consistent decreases in the activity of ChAT in aged rats were found, and the location of the changes was dependent on the sex of the rat. Aged female rats showed approximately 30% lower ChAT and 40% lower acetylcholinesterase (AChE) activity in the ventral globus pallidus (vGP). Aged males did not show decreased ChAT in the vGP but activity in the medial aspect of the horizontal diagonal band nucleus was 50% lower than in the young males. ChAT activity in four other closely aligned basal forebrain nuclei was not different between the young and aged rats. Analysis of cell number, density and area in the vGP by AChE histochemistry showed no significant differences between aged and young females. In addition, age and sex-dependent changes were measured in pituitary glucose-6-phosphate dehydrogenase activity. The relationship of the changes to age-dependent decrements in memory, the possible influence of gonadal hormones on aging, and the mechanisms responsible for age-related declines in ChAT activity are discussed.