Abstract The influence of aging on the acetylcholine synthesising and the degrading enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) was studied in the hippocampus of male Wistar rats at 2 months (young), 12 months (adult) and 27 months (old) of age using biochemical, immunocytochemical and histochemical techniques. The influence of treatment for 6 months with a daily dose of 100 mg/kg of choline alphoscerate ( l-α-glycerylphosphorylcholine) on the parameters examined was also investigated in old rats. Biochemical analysis of ChAT and AChE revealed the highest of the enzymatic activities in the hippocampus of adult rats and no significant differences between young and old animals. Immunocytochemical analysis of ChAT immunoreactivity revealed the highest immunostaining in adult rats followed in descending order by young then old animals. Histochemical evaluation of AChE reactivity revealed the highest expression in adult rats followed in descending order by old then young animals. Biochemical analysis of the effects of choline alphoscerate did not reveal any effect on ChAT activity and an increased expression of AChE activity. Moreover, the compound restored, in part, ChAT immunoreactivity in the hippocampus of old rats and increased the expression of AChE reactivity primarily in the CA 3 sub field in old rats. The above results suggest that appropriate quantitative immunocytochemical and histochemical techniques may represent a useful tool for assessing age-dependent changes in cholinergic neurotransmission markers. The functional and pharmacological significance of the effects of choline alphoscerate on the expression of ChAT and AChE in the hippocampus of aged rats should be clarified in future studies.