Whole cell clamp experiments were used to study the mechanism of action of amiloride on cardiac T-type Ca channels in guinea pig ventricular myocytes. Two hundred fifty microM amiloride blocked the T-type Ca channel for approximately 50%, whereas this concentration did not suppress the L-type Ca channel. The Kd value for the block of the T-type Ca channel was 233 microM and the Hill coefficient was 1.1. The drug showed a rapid onset of action and a quick wash out with complete reversibility. Amiloride blocked the T-type Ca channel without frequency-dependency, i.e., block was established at the holding potential (-90 mV) without being dependent on activation and inactivation of the channel. Amiloride reduced the amplitude of the T-type Ca current at all potentials without changing steady-state activation and inactivation. Amiloride did not affect the T-type Ca current when the drug was applied intracellularly via the pipette solution. In summary, amiloride blocked the T-type Ca channel in a channel state- and voltage-independent way.