TRPM7 channel kinase is a protein highly expressed in cells of hematopoietic lineage, such as lymphocytes. Studies performed in native and heterologous expression systems have shown that TRPM7 forms nonselective cation channels functional in the plasma membrane and activated on depletion of cellular Mg(2+). In addition to internal Mg(2+), cytosolic pH and the phospholipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] are potent physiological regulators of this channel: protons inhibit, while PI(4,5)P(2) is required for TRPM7 channel activity. These channels are also inhibited from inside by other metal cations and polyamines. While the regulation of TRPM7 channels by internal metal ions, acidic pH, and PI(4,5)P(2) is voltage independent, extracellular metal cations and polyamines block voltage dependently at micromolar concentrations and appear to occupy a distinct blocking site. In the present study we investigated intracellular Mg(2+) and pH dependence of native TRPM7 currents using whole cell patch-clamp electrophysiology in human Jurkat T lymphocytes and HEK293 cells. Our main findings are 1) Mg(2+) inhibition involves not one but two separate sites of high (∼10 μM) and low (∼165 μM) affinity; and 2) while sharing certain characteristics with Mg(2+) inhibition, protons most likely inhibit through one inhibitory site, corresponding to the low-affinity Mg(2+) site, with an estimated IC(50) of pH 6.3. Additionally, we present data on amplitude distribution of preactivated TRPM7 currents in Jurkat T lymphocytes in the absence of prior Mg(2+) or proton depletion.