Effects of changing cytosolic free Mg2+ concentration on L-type Ca2+ (I ) and Ba2+Ca currents (IBa) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 mM or 1.8 mM [Mg2+] ([Mg2+]p) buffered with 30 mM citrate acid and 10 mM ATP. Increasing [Mg2+]p from 0.2 mM to 1.8 mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (IT) protocols, respectively. Increasing [Mg2+]p shifted the VM for half inactivation by -20 mV but dramatically decreased ICa amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of IT amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg2+] might also inhibit permeation through open Ca2+p channels at positive VM.