The recent development of peptide antagonists that selectively block subtypes of neuronal calcium channel has provided tools to study the role of presynaptic calcium channels in triggering exocytosis. A variety of methods have consistently demonstrated that multiple channel types participate in exocytosis. We have studied the subsecond kinetics of [3H]glutamate release from rat cortical synaptosomes as an assay for presynaptic calcium channel activity. The system has been characterized over a broad range of conditions in an effort to compare biochemical measurements of transmitter release with electrophysiological measurements of synaptic currents. The efficacies of omega-agatoxin IVA and omega-conotoxins GVIA and MVIIC were increased when Ca2+ influx was decreased by: (1) decreasing the KCl concentration to diminish the extent of depolarization, (2) decreasing the Ca2+ concentration, or (3) partially blocking Ca2+ influx with one of the other antagonists. By using these toxins in combination, we found that at least three types of pharmacologically distinct channel participate in exocytosis. The largest fraction of glutamate release is blocked by omega-agatoxin IVA (IC50 = 12.2 nM) and by omega-conotoxin MVIIC (IC50 = 35 nM), consistent with the pharmacology of a P type channel. The effects of saturating concentrations (1 microM) of omega-agatoxin IVA or omega-conotoxin MVIIC occlude each other, suggesting that these peptides overlap completely. The specific N type antagonist omega-conotoxin GVIA inhibits a significant portion of release (IC50 less than 1 nM) but only under conditions of reduced Ca2+ concentration. These results suggest that the N type channel in nerve terminals is distinct from that found in hippocampal somata, since it appears to be resistant to by omega-conotoxin MVIIC. The combination of omega-conotoxin GVIA (100 nM) and either omega-agatoxin IVA or omega-conotoxin MVIIC (1 microM each) blocked approx 90% of release when the Ca2+ concentration was reduced (0.46 mM or less), but 30-40% of release remained when the concentration of Ca2+ in the stimulus buffer was 1 mM or greater, indicating that a resistant channel type(s) also participates in exocytosis. Specific inhibitors of this resistant phenotype will be useful for further refinement of our understanding of the role of presynaptic calcium channels in mediating neurosecretion.