Venoms from spiders are an important source of toxins that can help on the dissection of mechanisms involved in neurotransmission. Among them is the venom of the spider Phoneutria nigriventer that contains several toxic fractions with different targets in mammals and/or insects. We here report that one of these fractions (PhTx2) is able to evoke acetylcholine release from rat cortical synaptosomes and that this effect is dependent on extracellular calcium and is inhibited by the sodium channel blocker tetrodotoxin.