The effect of 20 mM extracellularly applied 2,3-Butanedione monoxime (BDM) on L-type Ca2+ channel charge movement current was studied in whole-cell voltage-clamped guinea-pig ventricular myocytes. Intramembraneous charge movement in response to depolarizing pulses (charge 1), was reduced after the application of BDM. The effect was more pronounced at the OFF of the charge transient (41%) than at the ON (7%). The steady-state availability curve of charge 1 was shifted to the left; the magnitude of the voltage shift was similar to the shift in Ca2+ current availability. Charge movement recorded in the negative voltage range (charge 2) after conditioning depolarizing pulses of different duration, was increased by BDM. For a 300-ms conditioning pulse charge 2 measured during a negative test pulse increased 40% (in Ba2+ external solution) or 35% (in Ca2+ external solution). These results show that BDM promotes voltage-dependent inactivation of L-type Ca2+ channels in parallel with charge interconversion between intramembranous charges 1 and 2. Mechanistically they are consistent either with dephosphorylation or a dihydropyridine-like action, but argue against open channel block as the mechanism of the effect of the drug.