The TRPM3, a member of the melastatin subfamily of transient receptor potential (TRP) ion channels, is a Ca²⁺ permeable non-selective cation channel activated by the neurosteroid pregnenolone sulphate (PS) and by heat. Recent studies reported the expression and functional role of TRPM3 in various tissues including pancreatic b-cells and sensory neurons, but the molecular regulation of the channel remains poorly understood. Intracellular ATP and PIP2 were shown to modulate several members of TRP family, but we lack any data regarding TRPM3. Therefore, we aimed to investigate the influence of intracellular factors on the activity of TRPM3. We carried out voltage clamp measurements using the cell-attached and inside-out configuration of the patch clamp technique on HEK293T cells overexpressing mouse TRPM3. The channels of the clamped membrane patches were stimulated by 100 mM PS applied to the extracellular side of the membrane via the pipette solution. In cell attached configuration, we measured moderate channel activity, which was dramatically increased in inside-out configuration just after the excision of the membrane patch. Current potentiation after excision was followed by rapid current decay. Application of 2 mM ATP to the cytosolic side of the inside-out membrane patch restored the TRPM3 activity. Kinetic analysis of the effect of cytosolic ATP indicated a dual effect on TRPM3: direct channel inhibition, which may be due to direct binding to the channel, and slow restoration of channel activity, which may represent the action of an ATP-dependent enzyme. Supporting the above hypothesis, the non-hydrolyzable ATP analog adenosine-5'-[(b,g)-methyleno] triphosphate (APPCP) applied to the cytosolic side of the membrane patch, in contrast to ATP, was failed to restore the channel activity. Our additional finding that PIP2 also caused a partial recovery of TRPM3 current in inside-out patches, suggests that ATP may act, at least partly, by fuelling the restoration of PIP2 levels in the plasma membrane. In our running experiments we are further exploring the molecular basis of the ATP-dependent TRPM3 regulation.