The magnesium-inhibited cation (MIC) current (I_MIC) in cardiac myocytes biophysically resembles currents of heterologously expressed transient receptor potential (TRP) channels, particularly TRPM6 and TRPM7, known to be important in Mg²⁺ homeostasis. To understand the regulation of MIC channels in cardiac cells, we used the whole-cell voltage-clamp technique to investigate the role of intracellular ATP in pig, rat and guinea-pig isolated ventricular myocytes. I_MIC, studied in the presence or absence of extracellular divalent cations, was sustained for 50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells, I_MIC exhibited complete run-down. Equimolar substitution of internal ATP by its non-hydrolysable analogue AMP-PNP failed to prevent run-down. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate (FVPP) prevented I_MIC run-down. In contrast, under similar conditions, neither the inhibition of protein phosphatases 1, 2A, 2B, or of protein tyrosine phosphatase, nor the activation of protein kinase A (forskolin, 20 µM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent run-down. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) by preventing its re-synthesis (wortmannin, 10 µM, or phenylarsine oxide, 15 µM) induced run-down of I_MIC. Finally, loading ATP-depleted cells with exogenous PIP₂ (10 µM) prevented run-down. These results suggest that PIP₂, likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity.