Involvement of P-type voltage-dependent Ca2+ channels in spinal morphine- or clonidine-induced antinociception and in the synergistic interaction between morphine and clonidine was examined in the present studies. Coadministration of the selective P-type antagonist, omega-agatoxin IVA (25 ng) intrathecally (i.t.) to mice along with morphine or clonidine enhanced the tail flick antinociception of each agonist 5-6-fold. The greater-than-additive (synergistic) interaction that occurred when morphine and clonidine were coadministered i.t. decreased to an additive interaction in the presence of omega-agatoxin IVA. In mice pretreated with pertussis toxin (10 ng) to inactivate G proteins, omega-agatoxin IVA did not alter the morphine/clonidine synergism. Surprisingly, omega-agatoxin IVA reversed the additive morphine/clonidine interaction that occurs in morphine-tolerant mice back to synergism. These results suggest that functional P-type Ca2+ channels play an essential role in the antinociceptive synergism between spinal morphine and clonidine.