PURPOSE OF STUDY: In VSMCs, Ca-activated Cl channels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). The mechanisms by which ANO1 influence the excitability of VSMCs remains to be elucidated due to questionable pharmacology and lack of a reliable genetic knockdown mouse model of ANO1.The aim of this study was to re-evaluate the role of ANO1 in electromechanical coupling of pulmonary artery (PA) smooth muscle using newer generation ANO1 blockers and a novel smooth muscle-specific inducible ANO1 knockout mouse model (SMC-iANO1-KO). METHODS USED: Wire myography was used to determine the vascular reactivity to 5-HT of PA from wild-type and SMC-iANO1-KO mice. Calcium imaging experiments were also carried out using SMC-iGCaMP3 mice, which genetically express the Ca biosensor GCaMP3 in smooth muscle cells. SUMMARY OF RESULTS: 5-HT elicited a dose-dependent contraction (0.01–30 µM) that was similarly inhibited (~50%–70%) by the ANO1 blocker CaCCInh-A01 (10 µM), the CaV1.2 blocker nifedipine (1 µM) or the SERCA2 pump inhibitor cyclopiazonic acid (CPA; 10 µM). Genetic ablation of ANO1 produced a reduction in 5-HT-induced tone (~60% at 1 µM 5-HT) that was similar to that produced by CaCCinhA01, nifedipine or CPA. Ca imaging experiments in the intact PA of SMC-iGCaMP3 mice revealed that 5-HT evoked spatially and temporally localised Ca transients. These Ca oscillations were potently inhibited by CaCCInh-A01 or nifedipine, and were abolished by CPA. CONCLUSIONS: In conclusion, 5-HT elicited highly localised Ca oscillations that were promoted by Ca entry through CaV1.2, most likely involving transient depolarizations evoked by ANO1 activated by a balance between oscillatory SR Ca release through IP3 receptors and Ca entry through CaV1.2. We propose that the stable agonist-induced PA contraction results from the integration of stochastic and localised Ca events supported by a microenvironment comprising ANO1, CaV1.2 and IP3 receptors.