Peptide channel blockers found in venoms of many predators are useful pharmacological tools and potential therapeutic agents. The venom of the Brazilian spider Phoneutria nigriventer contains a fraction, -Phonetoxin-IIA (-Ptx-IIA, 8360 MW), which blocks Ca²⁺ channels. At frog neuromuscular junctions (nmj) bathed in normal Ca²⁺ (1.8 mM) saline, -Ptx IIA did not affect spontaneous transmitter release but reversibly reduced evoked transmitter release by 75% and 95% at 12 and 24 nM, respectively. In contrast, toxin effects were irreversible in low Ca²⁺ (0.5 mM) saline. Ca²⁺ imaging in normal Ca²⁺ saline showed that -Ptx-IIA partially blocked stimulus-dependent presynaptic Ca²⁺ signals and the blockade was almost completely reversible. Increases in spontaneous release frequency induced by high extracellular K⁺ were blocked by -Ptx-IIA. Therefore -Ptx-IIA blocks N-type Ca²⁺ channels which admit Ca²⁺ that triggers transmitter release at the frog nmj. Additional evidence predicts that -Ptx-IIA binds to N-type Ca²⁺ channels at a different site than -Conotoxin-GVIA. -Ptx-IIA also gave a low affinity partial blockade of transmitter release and presynaptic Ca²⁺ signals at crayfish nmjs where P-type channels are blocked by -Agatoxin-IVA. The Ca²⁺ dependent reversibility and promiscuity of this toxin may make it highly useful experimentally and therapeutically.