To elucidate the types of voltage-dependent Ca²⁺ channels controlling acetylcholine and catecholamine releases in the in vivo adrenal medulla, we implanted microdialysis probes in the left adrenal medulla of anesthetized rats and investigated the effects of Ca²⁺ channel antagonists on acetylcholine, norepinephrine, and epinephrine releases induced by nerve stimulation. The dialysis probes were perfused with Ringer's solution containing a cholinesterase inhibitor, neostigmine. The left splanchnic nerves were electrically stimulated at 2 and 4 Hz before and after intravenous administration of Ca²⁺ channel antagonists. -Conotoxin GVIA (an N-type Ca²⁺ channel antagonist, 10 µg/kg) inhibited acetylcholine release at 2 and 4 Hz by about 40%, norepinephrine release at 4 Hz by about 50%, and epinephrine release at 2 and 4 Hz by about 45%. A 5-fold higher dose of -conotoxin GVIA (50 µg/kg) did not further inhibit these releases. -Conotoxin MVIIC (a P/Q-type Ca²⁺ channel antagonist, 50 µg/kg) inhibited acetylcholine and epinephrine releases at 4 Hz by about 30%. Combined -conotoxin GVIA (50 µg/kg) and MVIIC (250 µg/kg) inhibited acetylcholine release at 2 and 4 Hz by about 70%, norepinephrine and epinephrine releases at 2 and 4 Hz by about 80%. Nifedipine (an L-type Ca²⁺ channel antagonist, 300 and 900 µg/kg) did not change acetylcholine release at 2 and 4 Hz, but nifedipine (300 µg/kg) inhibited epinephrine release at 4 Hz by 20% and nifedipine (900 µg/kg) inhibited norepinephrine and epinephrine releases at 4 Hz by 30%. In conclusion, both N- and P/Q-type Ca²⁺ channels control acetylcholine release on pre-ganglionic splanchnic nerve endings while L-type Ca²⁺ channels do not. L-type Ca²⁺ channels are involved in norepinephrine and epinephrine releases on chromaffin cells.