The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.