The sensitivity of synaptic transmission to antagonists of different calcium channels was examined at the powerful climbing fiber synapse between neurons from the inferior olive and cerebellar Purkinje cells. In rat brain slices, climbing fibers were activated with extracellular electrodes, and synaptic currents were recorded with whole-cell patch clamp. Dihydropyridines did not discernibly affect synaptic strength. omega-Conotoxin GVIA, a potent antagonist of N-type calcium channels, reduced synaptic currents by an average of 29%. omega-Agatoxin-IVA, a high affinity blocker of P-type calcium channels, reduced synaptic strength by an average of 77%. Together, the two toxins virtually eliminated synaptic transmission (91% inhibition). These results indicate that omega-agatoxin-IVA-sensitive calcium channels play an important role in transmission at the climbing fiber synapse. They also suggest that in single climbing fibers, release is evoked by at least two pharmacologically distinct calcium currents, one sensitive to omega-agatoxin-IVA, the other to omega-conotoxin GVIA.