TRP cation channels are increasingly recognized as membrane ‘coincidence detectors’ of a number of activator stimuli. Using patch-clamp techniques we have investigated the effects of thapsigargin (TG, 1 μM) and common phospholipids – lysophosphatidylcholine (LPC, 10 µM) and lysophosphatidylinositol (LPI, 10 µM) on the gating of TRPM8 channel, which was cloned from human prostate and stably expressed in HEK-293 cells. The expressed channel protein behaved as a ‘classical’ cold receptor (e.g. activation by depolarization, cold, menthol and icilin). In the cell-attached configuration cation channel conductance was 73.8±3.6 pS (n=15); its open probability (NPo) was increased upon cooling (t_1/2=26.6±0.9°C, n=5) from NPo=0.01±0.005 at 37°C (n=6) to NPo=0.26±0.05 at 20°C (n=15) at 80 or 100 mV. Furthermore, NPo voltage dependence was similar to that of cold- or menthol-induced whole-cell current. Thus, we have identified the 74 pS channel activity as a functional counterpart of the expressed TRPM8 protein. Strikingly, channel activity was also potentiated by TG in a time-dependent manner (NPo was 0.12±0.02, 0.46±0.11 and 1.37±0.18 in control, 2 and 10 min after TG application, respectively; n=10). Since iPLA₂, a Ca²⁺-independent cytosolic enzyme which produces an array of lysophospholipids, is up-regulated by Ca²⁺ store depletion (Smani et al. 2004) we next tested its possible involvement in TRPM8 activation. The effect of TG on TRPM8 activity was abolished following treatment with the iPLA₂ blocker bromoenol lactone. Testing two main iPLA₂ products, LPC and LPI, on single channel gating in inside-out patches revealed the underlying channel mechanisms. Channel activity was strongly reduced upon patch excision (4.6±0.7-fold in cell-attached vs. inside-out, n=13) but application of either LPC or LPI to the cytoplasmic side not only restored the activity (as was the case for PIP₂ action, Liu & Qin, 2005) but significantly increased NPo (from 0.03±0.01 in control to 0.20±0.09 or 0.93±0.33 with LPC or LPI, respectively; n=5-7). This potentiating effect was associated with the appearance of long channel openings which were also seen during the TG action. These long openings with the mean dwell time of 7.9±2.4 ms and relative contribution of 39±9% (n=7) generated >90% of the mean current. In contrast, in control channel openings were brief (0.48±0.03 ms at 80 mV) while longer openings were only occasionally observed at a very strong depolarization (2.92±1.44 ms, 1.3% contribution at 120 mV in 3 out of 12 patches). Even after menthol application their contribution was only 6±4% although they became more readily detectable (6/6 patches). Thus, we found that physiologically relevant phospholipids can stabilize open conformation of TRPM8 channel far better than other known activating factors.