To evaluate whether any degenerative changes affect the brain cholinergic systems during natural aging, we compared various cholinergic biochemical markers (number of muscarinic receptors, mAChR; choline acetyltransferase activity, ChAT; acetylcholinesterase activity, AChE; and sodium-dependent high affinity choline uptake) in the cortical (CR) and subcortical (SS) regions of the brains of aged (24 month) and young (2 month) rats. Using [3H]-quinuclidinyl benzilate ([3H]-QNB) as the ligand of muscarinic receptor binding, the numbers of mAChR decreased about 30% in both the CR and the SS of aged rats compared with those in young rats, while a significant age-related increase in the affinity of mAChR was observed. [3H]-QNB binding in both the young and aged rat brain was displaced markedly by pirenzepine, while [3H]-QNB binding in the SS of the aged rat brain was displaced at low concentrations of atropine. The Vmax values of ChAT and AChE also decreased about 20-30% compared with those of young rats. The sodium-dependent high affinity choline uptake was lower in the crude synaptosomal fraction prepared from aged rat brain than in young brain. Hemicholinium-3 inhibited the choline uptake in young rat brain at a concentration range of 1 microM-10 nM, but choline uptake in aged brain was insensitive to hemicholinium-3. These results indicate that natural aging brings about a diffuse and multiple depletion of various biochemical markers in cholinergic neurons.