Transient receptor potential melastatin 7 (TRPM7) channels were originally identified electrophysiologically when depletion of cytosolic Mg(2+) resulted in the gradual development of an outwardly rectifying cation current. Conversely, inclusion of millimolar Mg(2+) in internal solutions prevented activation of these channels in whole cell patch clamp. We recently demonstrated that the Jurkat T-cell whole cell TRPM7 channels are inhibited by internal Mg(2+) in a biphasic manner, displaying high [IC(50(1)) ≈ 10 μM] and low [IC(50(2)) ≈ 165 μM] affinity inhibitor sites. In that study, we had characterized the dependence of the maximum cell current density on intracellular Mg(2+) concentration. To characterize Mg(2+) inhibition in Jurkat T cells in more detail and compare it to whole cell results, we recorded single TRPM7 channels in cell-free membrane patches and investigated the dependence of their activity on Mg(2+) added on the cytoplasmic side. We systematically varied free Mg(2+) from 265 nM to 407 μM and evaluated the extent of channel inhibition in inside-out patch for 58 patches. We found that the TRPM7 channel shows two conductance levels of 39.0 pS (γ(1)) and 18.6 pS (γ(2)) and that both are reversibly inhibited by internal Mg(2+). The 39.0-pS conductance is the dominant state of the channel, observed most frequently in this recording configuration. The dose-response relation in inside-out patches shows a steeper Mg(2+) dependence than in whole cell, yielding IC(50(1)) of 25.1 μM and IC(50(2)) of 91.2 μM.. Single-channel analysis shows that the primary effect of Mg(2+) in multichannel patches is a reversible reduction of the number of conducting channels (N(o)). Additionally, at high Mg(2+) concentrations, we observed a saturating 20% reduction in unitary conductance (γ(1)). Thus Mg(2+) inhibition in whole cell can be explained by a drop in individual participating channels and a modest reduction in conductance. We also found that TRPM7 channels in some patches were not sensitive to this ion at submaximal Mg(2+) concentrations. Interestingly, Mg(2+) inhibition showed the property of use dependence: with repeated applications, Mg(2+) effect became gradually more potent, which suggests that Mg(2+) sensitivity of the channel is a dynamic characteristic that depends on other membrane factors.