Introduction. Ca2+-activated chloride channels (ClCa) are important excitatory mechanism in vascular myocytes because activation of ClCa leads to Cl- ion efflux and membrane depolarisation sufficient to enhance voltage gated Ca2+ channels. Our lab recently showed that the amplitude and pharmacology of ClCa currents (IClca) in portal vein myocytes was influenced by lipid raft integrity (1). The present study aimed to ascertain whether IClca in rat pulmonary arteries (PA) myocytes were also regulated by cholesterol as well as phosphatidylinositol 4, 5-bisphosphate (PIP2) levels. Methods. Whole-cell configuration of the patch clamp technique was used to measure IClca currents in myocytes dispersed from first and secondary-order branches of PA, from male Wistar rats (200-225g). The pipette solution contained a known amount of free Ca2+ (25-500 nM) which immediately induced an IClca upon rupture of the cell membrane. Upon stabilisation of the IClca, current voltage (IV) relationships were constructed by stepping from a holding potential (-50mV) to test potentials between -100 mV and 100 mV for 1s. All experiments was recorded after a control IV with 500nM [Ca2+]i, apart from wortmannin which was pre-incubated with the cells for 1hour, here untreated myocytes IClca was recorded on the same day. IClca was normalised to cell capacitance. Results. IClca in rat PA were activated in a Ca2+-dependent manner and displayed the distinctive voltage-dependent kinetics and anionic permeability profile of previous studies. Application of niflumic acid (100 μM) produced a characteristic complex effect whereas the novel TMEM16A inhibitor T16Ainh-AO1 (5 μM) produced simple inhibition. IClca was augmented rapidly by the cholesterol depleting agent methyl-β-cyclodextrin (3mg/ml) and after 1 h incubation with the phosphatidylinositol 4-kinase inhibitor wortmannin (20 μM) (~ 73.5% and 80.8% respectively, n=5). Conversely, the α1-adernergic receptor agonist methoxamine (1 μM) attenuated the IClca by ~ 32% (n=3). Conclusion. This study demonstrates that IClca are augmented by cholesterol depletion from the cell membrane and reduction of PIP2. An augmented IClca in a response to a reduction in levels of PIP2 suggests that PIP2 has an inhibitor function on the channel. However, activation of Gq linked α1-adernergic receptors produced a marked reduction in IClca suggesting the regulation of ClCa by PIP2 is complicated.