The effects of kamikihi-to (EK-49) on various cholinergic biochemical markers, muscarinic receptors, acetylcholinesterase (AChE), choline acetyltransferase (CAT) and choline uptake, in aged rat brains were examined. Administration of kamikihi-to in daily doses of 200 mg/kg, p.o. for 4 weeks to aged rats (24-months-old) significantly increased the density of muscarinic receptors (Bmax) for 3H-QNB (quinuclidinyl benzilate), but did not affect the apparent dissociation constant (Kd). After repeated administration of kamikihi-to to aged rats, AChE activity and choline uptake were not significantly different in the kamikihi-to treated aged rats as compared with the control aged rats. However, administration of kamikihi-to for 4 weeks resulted in a significant increase in Vmax values of CAT in kamikihi-to treated aged rats as compared with those of the control rats. These results indicate that long-term treatment with kamikihi-to enhances both the Bmax and Vmax for QNB binding and CAT activity in aged rats, respectively.