The venom of the Brazilian spider Phoneutria nigriventer possesses several neurotoxic polypeptidic fractions. Previous work has established that one of the toxic components, PhTx3, inhibited Ca(2+)-dependent glutamate release and the increase in cytosolic free Ca2+ in response to membrane depolarization. In the present work, we investigated the effect of PhTx3 on the release of acetylcholine (ACh) from brain and peripheral neurons. PhTx3 decreased the release of [3H]-ACh induced by tityustoxin and KCl in brain cortical slices and myenteric plexus. The inhibitory effect of myenteric plexus had the same magnitude as that obtained in the absence of extracellular Ca2+. However, in brain PhTx3 was less efficient at decreasing the evoked release of ACh. These experiments suggest that the target of PhTx3 is coupled to the process of release of ACh in brain and autonomic nervous system.