In this report we show that α-latrotoxin from black widow spider venom is a potent activator of neurotransmitter release in synaptosomes from the Torpedo electric organ. Binding of the purified toxin (5 nM) to the synaptosomal fraction occurs already at 4°C and is dependent on the presence of divalent ions. However, neurotransmitter release commences only after temperature elevation (22°C) and is completed within 2 min. The effect of α-latrotoxin on release is achieved at 1 nM and is already saturated at 5 nM. The release is stimulated by the presence of Ca2+ ions. Activation of release by α-latrotoxin is accompanied by morphological changes in electric organ synaptosomes. The synaptosomes swell, resulting in a 55% increase in section area. Moreover, the number of synaptic vesicles per unit area decreases about three-fold, and rows of docked synaptic vesicles are rarely detected as opposed to control synaptosomes. These morphological changes indicate that the massive release is mainly due to synaptic vesicle fusion. α-Latrotoxin binding sites are highly concentrated in the innervated face of the electrocytes. lmmunoelectron microscopy on electric organ sections reveals alatrotoxin binding sites over the entire plasma membrane at release sites and facing Schwann cells surrounding Torpedo nerve terminals. Surprisingly, a high concentration of binding sites is also found at structures surrounding branching unmyelinated axons. This staining is in close proximity to Schwann cell envelopes and to the basal lamina around axonal tips. The mode of action of α-latrotoxin in view of the localization of its binding sites is discussed.