Presynaptic calcium channels are crucial elements of neuronal excitation-secretion coupling. In mammalian brain, they have been difficult to characterize because most presynaptic terminals are too small to probe with electrodes, and available pharmacological tools such as dihydropyridines and ω-conotoxin are largely ineffective. Subsecond measurements of synaptosomal glutamate release have now been used to assess presynaptic calcium channel activity in order to study the action of peptide toxins from the venom of the funnel web spider Agelenopsis aperta, which is known to inhibit dihydropyridine and ω-conotoxin-resistant neuronal calcium currents. A presynaptic calcium channel important in glutamate release is shown to be ω-Aga-IVA sensitive and ω-conotoxin resistant.