The potential for calcium-activated chloride channels (CaCCs) as drug targets has led to the recent development of novel specific small molecule inhibitors of them [1]. These drugs are also enabling a much improved pharmacological dissection of the role of CaCCs within tissues. Myometrial contractions need to be regulated for successful pregnancy and parturition. Our previous electrophysiological studies showed CaCCs in 30% of isolated myometrial cells in pregnant rats [2]. The aims of this study are thus to use these new inhibitors of CaCCs to determine the functional effects of CaCCs on human and rat myometrium and characterise the effect of CaCCs inhibition on Ca signaling. Strips of myometrium (4 x 1mm) were dissected from biopsies taken from term, pre-labour elective caesarean sections in women and labouring (LR) or term pregnant (TPR) rats. Contractility was measured on strips under isometric conditions in HEPES buffered physiological salt solution at 370C and in some experiments simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and force were made on strips previously loaded with Indo -1. The CaCC inhibitor (CaCCinh-A01) and TMEM16A inhibitor (T16Ainh-A01) were both used at 30µM for 10-20 min based on preliminary dose response experiments. Data are mean ± S.E.M. and changes in baseline [Ca2+]i are normalised to the amplitude of control phasic transients (100%). Control spontaneous phasic contractions were established for 30 – 60 min. CaCCinh-A01 completely inhibited spontaneous contractions in all myometrial strips (human, n=5; LR, n=2 and TPR n=3. In contrast T16Ainh-A01 had no effect on spontaneous activity in any preparation. In rats during control contractions, spontaneous phasic Ca transients (Ca entry through voltage-gated Ca channels; VOCCs) were recorded and basal Ca remained stable. The CaCCinh-A01 induced an immediate and profound decrease in basal [Ca2+]i before cessation of spontaneous Ca and force transients. After 10 min basal [Ca2+]i had decreased by 52 ±2%, n=4. CaCC inhibition has been shown to hyperpolarise smooth muscle [3] which might inhibit Ca entry through VOCCs and thus decrease basal Ca. Experiments were therefore repeated in nifedipine (an inhibitor of VOCCs). Nifedipine (1µM) inhibited spontaneous Ca and force transients and decreased basal [Ca2+]i by 15%. However CaCCinh-A01 further decreased basal Ca2+]i by 40% (preliminary data, n=2) suggesting that a significant component of CaCC activity on [Ca2+]i is independent of VOCC entry. These data suggest that there is a critical and constitutive role for CaCCs, but not the specific TMEM16A channel, in the myometrium in maintaining basal [Ca2+]i and spontaneous activity. Further work using confocal microscopy will help elucidate the precise role of CaCCs on local and global intracellular calcium.