Cortex and periaqueductal grey (PAG) play a major role in the pathophysiology of migraine. Some antiepileptic drugs (AEDs) influence the activity of these structures by modulating high-voltage-activated (HVA) Ca2+ channels and are effective in migraine prevention. The aim of the present study was to investigate the expression of total HVA Ca2+ channels in cortical and PAG neurons and to study the differential action of AEDs on these channels. Isolated neurons were visually identified based on morphological criteria. HVA currents were recorded by whole-cell patch-clamp technique. The distribution ratio of L-, N-, P-, Q- and R-type HVA Ca2+ channels was different between cortical and PAG neurons. In particular, we found that P- and Q-type HVA Ca2+ channels were more expressed in PAG neurons than in cortical cells, whereas L- and R-type HVA Ca2+ channels showed an opposite distribution. Interestingly, N-type HVA Ca2+ channels were equally distributed in these two neuronal populations. A differential sensitivity to AEDs of HVA Ca2+ channels located on cortical and PAG neurons was observed for topiramate (TPM), but not for lamotrigine (LTG) or levetiracetam (LEV). In fact, whereas both LTG and LEV were equally effective and potent in inhibiting HVA Ca2+ currents in the two neuronal populations, TPM showed a much higher potency and efficacy in blocking these currents in PAG neurons than in cortical pyramidal cells. TPM, in fact, inhibited N-, P- and L-type channels in PAG neurons, whereas in cortical neurons this AED modulated only P- and L-type channels. Unlike the other AEDs investigated, valproic acid did not affect HVA Ca2+ currents in cortical and PAG neurons. The negative modulation of specific subtypes of HVA Ca2+ channels by various AEDs can restore normal electrical activity in target brain areas such as cortex and PAG, providing interesting therapeutic approaches in migraine prevention.