Agatoxins from Agelenopsis aperta venom target three classes of ion channels, including transmitter-activated cation channels, voltage-activated sodium channels, and voltage-activated calcium channels. The alpha-agatoxins are non-competitive, use-dependent antagonists of glutamate receptor channels, and produce rapid but reversible paralysis in insect prey. Their actions are facilitated by the micro-agatoxins, which shift voltage-dependent activation of neuronal sodium channels to more negative potentials, causing spontaneous transmitter release and repetitive action potentials. The omega-agatoxins target neuronal calcium channels, modifying their properties in distinct ways, either through gating modification (omega-Aga-IVA) or by reduction of unitary current (omega-Aga-IIIA). The alpha-agatoxins and omega-agatoxins modify both insect and vertebrate ion channels, while the micro-agatoxins are selective for insect channels. Agatoxins have been used as selective pharmacological probes for characterization of ion channels in the brain and heart, and have been evaluated as candidate biopesticides.