The neurotransmitter of the cholinergic system, acetylcholine plays a major role in the brain's cognitive function and is involved in neurodegenerative disorders. Here, we present age-related alterations of acetylcholine levels after administration of the acetylcholinesterase inhibitor drug tacrine in normal mice. Using a quantitative, robust and molecular-specific mass spectrometry imaging method we found that tacrine administration significantly raised acetylcholine levels in most areas of sectioned mice brains, inter alia the striatum, hippocampus and cortical areas. However, acetylcholine levels in retrosplenial cortex were significantly lower in 14-month-old than in 12-week-old animals following its administration, indicating that normal aging affects the cholinergic system's responsivity. This small brain region is interconnected with an array of brain networks and is involved in numerous cognitive tasks. Simultaneous visualization of distributions of tacrine and its hydroxylated metabolites in the brain revealed a significant decrease in levels of the metabolites in the 14-month-old mice. The results highlight strengths of the imaging technique to simultaneously investigate multiple molecular species and the drug-target effects in specific regions of the brain. The proposed approach has high potential in studies of neuropathological conditions and responses to neuroactive treatments.