Background: Ca2+-activated Cl- channels (CaCCs) are thought to be encoded for by the gene TMEM16A, and are functionally present in vascular and non-vascular smooth muscle cells (Davis et al., 2010, Davis et al., 2013). Our lab recently showed that the amplitude and pharmacology of CaCC currents (ICaCC) in portal vein myocytes was influenced by cholesterol depletion (Sones et al., 2010). The aim of this study was to investigate the mechanism by which CaCCs were regulated by cholesterol depletion and the possible role of phosphatidylinositol 4, 5-bisphosphate (PIP2) levels. Methods and Results: Whole-cell configuration of the patch clamp technique was used to measure ICaCC currents in myocytes dispersed from first and secondary-order branches of pulmonary arteries (PA), from male Wistar rats (200-225g), killed by cervical dislocation in accordance with Schedule 1 of the United Kingdom Animals Act (1986). The pipette solution contained a known amount of free Ca2+ (500 nM) which immediately induced an ICaCC upon rupture of the cell membrane, which displayed the distinct voltage-dependent kinetics shown in previous studies. Upon stabilisation of the ICaCC, current voltage (IV) relationships were constructed by stepping from a holding potential (-50mV) to test potentials between -100 mV and 100 mV for 1s. ICaCC was augmented rapidly by the cholesterol depleting agent methyl-β-cyclodextrin (mβcd) (3mg/ml), which was prevented by the application of the TMEM16A inhibitor T16Ainh-A01. Cells pre-treated with the phosphatidylinositol 4-kinase inhibitor wortmannin (20 μM), thereby inhibiting PIP2 synthesis, augmented ICaCC and subsequently showed an attenuated response to mβcd application. As wortmannin is also a myosin light chain kinase (MLCK) inhibitor we tested ML-9 (10 µM) to establish if MLCK played a role in CaCC activity, but this had no effect on the current. Enrichment of the pipette solution with diC-8 PIP2 (1 μM) attenuated ICaCC, and attenuated the stimulatory effect of mβcd. Conclusions: This study suggests that cholesterol depletion of the cell membrane increases TMEM16A activity, partly through the removal on the inhibitory effect of PIP2.