It has been shown that mibefradil (Ro 40-5967) exerts a selective inhibitory effect on T-type Ca2+ currents, although at higher concentrations it can antagonize high voltage-activated Ca2+ currents. The action of mibefradil on Ca2+ channels is use- and steady-state-dependent and the binding site of mibefradil on L-type Ca2+ channels is different from that of dihydropyridines. By using conventional whole-cell and perforated patch-clamp techniques, we showed that mibefradil has an inhibitory effect on both T- and L-type Ca2+ currents in insulin-secreting cells. However, the effect on L-type Ca2+ currents was time-dependent and poorly reversible in perforated patch-clamp experiments. By using mass spectrometry, we demonstrated that mibefradil accumulates inside cells, and furthermore, a metabolite of mibefradil was detected. Intracellular application of this metabolite selectively blocked the L-type Ca2+ current, whereas mibefradil exerted no effect. This study demonstrates that mibefradil permeates into cells and is hydrolyzed to a metabolite that Blocks L-type Ca2+ channels specifically by acting at the inner side of the channel.