The effects of changing cytosolic [Mg2+] ([Mg2+]i) on L- type Ca2+ currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg2+] and [Ca2+]. To control [Mg2+]i and cytosolic [Ca2+] ([Ca2+] i), the pipette solution included 30 mM citrate and 10 mM ATP along with 5 mM EGTA (slow Ca2+ buffer) or 15 mM EGTA plus 5 mM BAPTA (fast Ca2+ buffer). With pipette [Ca2+] ([Ca2+]p) set at 100 nM using a slow Ca2+ buffer and pipette [Mg2+] ([Mg2+]p) set at 0.2 mM, peak L-type Ca2+ current density (ICa) was 17.0 ± 2.2 pA pF- 1. Under the same conditions, but with [Mg2+]p set to 1.8 mM, ICa was 5.6 ± 1.0 pA pF-1, a 64 & [plusmn] 2.8 % decrease in amplitude. This decrease in ICa was accompanied by an acceleration and a -8 mV shift in the voltage dependence of current inactivation. The [Mg2+]p-dependent decrease in ICa was not significantly different when myocytes were preincubated with 10 µM forskolin and 300 µM 3-isobutyl-1-methylxanthine and voltage-clamped with pipettes containing 50 µM okadiac acid, to maximize Ca2+ channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing Protein Phosphatase 2A, to promote channel dephosphorylation, ICa decreased only 25 & [plusmn] 3.4 % on changing [Mg2+]p from 0.2 to 1.8 mM. In the presence of 0.2 mM [Mg2+]p, changing channel phosphorylation conditions altered ICa over a 4-fold range; however, with [Mg2+]p = 1.8 mM, these same maneuvers had no significant effect on ICa. These data suggest that [Mg2+]i can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca2+]p to 1, 100 or 300 nM also showed that the [Mg2+]p- induced reduction of ICa was smaller at the lowest [Ca2+] p, irrespective of channel phosphorylation conditions. This interaction between [Ca2+]i and [Mg2+]i to modulate ICa was not significantly affected by ryanodine, fast Ca2+ buffers or inhibitors of calmodulin, calmodulin- dependent kinase and calcineurin. Thus, physiologically- relevant [Mg2+]i modulates ICa by counteracting the effects of Ca2+ channel phosphorylation and by an unknown [Ca2+]i-dependent mechanism. The magnitude of these effects suggest that changes in [Mg2+]i could be critical in regulating L-type channel gating.