The effect of prejunctional blocker Toosendanin (TSN) on acetylcholine (ACh) level in striatum and parietal cortex was investigated by means of method of brain dialysis coupled with microbore high-performance liquid chromatograph (HPLC)-electrochemical detection (ECD) with enzyme immobilized post-column at freely moving rats. The results are as follows: (1) TSN inhibits high K+ (100 mM)-induced ACh release from the mentioned two regions of brain, completely and irreversibly. (2) Before the inhibition, there appears a transient rise of ACh level. (3) This rise of ACh level can be blocked by TTX. (4) Calcium is necessary for the onset of this rise, but not for that of the sustained decrease of ACh level. These results indicate that the effect of TSN on central cholinergic synapses is similar to that of neuromuscular junction, namely, its blocking action is always preceded by a Ca(2+)-dependent facilitatory phase with result of a TSN-induced transient rise of ACh, but the sustained decline and final blockade of ACh release are Ca(2+)-independent.