Clinical observations and genetic studies have suggested a role for high-threshold voltage-dependent calcium channels (VDCCs) in the pathogenesis of migraine. This study investigated the role of P/Q-, L- and N-type VDCCs in post-synaptic action potential generation in trigeminovascular nociceptive afferents in the trigeminocervical complex (TCC) of the cat in vivo. Trigeminovascular nociceptive afferents were identified in the TCC by electrical stimulation of the superior sagittal sinus. Forty-six cell bodies were identified by their response to microiontophoresis of l-glutamate and their bipolar action potential shape. Blockade of VDCCs was accomplished by microiontophoresis of omega-agatoxin IVa/TK (P/Q-), omega-conotoxin GVIa (N-) and calciseptine (L-type). Non-selective antagonism was studied using cadmium ions. Non-selective blockade of high threshold VDCC with cadmium resulted in a reduction in l-glutamate-evoked neuronal activity (P=0.01). Blockade of P/Q: TK- (P<0.001), IVA- (P=0.007), L- (P<0.001) and N-type (P<0.001) VDCCs resulted in significant reductions in post-synaptic action potential generation in response to l-glutamate. High threshold VDCCs, including P/Q-, L- and N-type VDCCs, can therefore modulate nociceptive transmission in the trigeminocervical complex in vivo. We discuss the evidence to suggest a role for VDCCs in the pathophysiology of primary headache disorders, and how abnormalities of function may contribute to their pathogenesis.