In urethane-anaesthetised artificially ventilated Sprague–Dawley rats, bilateral microinjection of the divalent cation nickel chloride (Ni²⁺; 50 mM, 50 nl) into the rostral ventrolateral medulla elicited a dramatic inhibition of splanchnic sympathetic nerve activity (−44±6%) and a marked depressor response (−35±7 mmHg). Selective blockade of high-voltage activated Ca²⁺ channels with ω-agatoxin IVA (P/Q-type), ω-conotoxin GVIA (N-type) and nifedipine (L-type) did not decrease arterial pressure or splanchnic sympathetic nerve activity when injected separately into the rostral ventrolateral medulla, or combined with kynurenate. Injection of caesium chloride or ZD 7288, a blocker of the hyperpolarization-activated cation current, into the rostral ventrolateral medulla had no effect on arterial pressure or splanchnic sympathetic nerve activity. Bilateral microinjection of nickel chloride into the caudal ventrolateral medulla/pre-Bötzinger complex elicited small increases in splanchnic sypathetic nerve activity (+17±13%) and arterial pressure (+12±4 mmHg). These were substantially smaller than those evoked by blockade of glutamatergic receptors or high-voltage activated Ca²⁺ channels in this area. Injection of kynurenate or high-voltage activated Ca²⁺ channel blocker, but not Ni²⁺, in this area evoked respiratory termination. The results indicate the existence of a distinct mechanism maintaining the tonic activity of rostral ventrolateral medulla presympathetic neurons that is different from that maintaining the tonic activity in the caudal ventrolateral medulla/pre-Bötzinger region. We conclude that ion channels that are sensitive to Ni²⁺, but are insensitive to high-voltage activated (L, P/Q, N) Ca²⁺ channel blockers, and are located postsynaptically on the presympathetic rostral ventrolateral medulla neurons are responsible for the tonic activity of the presympathetic neurons in rostral ventrolateral medulla. These channels could well be the low-voltage-activated (or T-type) Ca²⁺ channels although other conductances cannot be conclusively excluded.