Several signalling components, including Ca channels are enriched in plasma membrane domains high in cholesterol; lipid rafts and caveolae. Signalling cascades maintain uterine quiescence during pregnancy until the onset of labour. Myometrial cholesterol is higher in pregnant that non-pregnant women and may be further elevated in obese mothers. Moreover, cholesterol manipulation in rat uterus destabilises lipid rafts and disrupts caveolae. We hypothesised that disruption of lipid domains following cholesterol manipulation may be detrimental to Ca signalling and reduce myometrial contractility, particularly in the obese mothers. Following ethical approval, uterine biopsies were obtained with consent from pregnant women undergoing Caesarean sections and non-pregnant women having hysterectomies. Cholesterol was loaded and extracted with water-soluble cholesterol (5mg/ml) and 2% methyl-B-cyclodextrin, respectively. The impact on force and intracellular [Ca] (Indo-1) were examined. Statistical significance (P<0.5) was determined using ANOVA and Tukeys test. The contractility of 9 non-pregnant samples significantly declined or was abolished by cholesterol. Significant decline in total force fell to a mean value of 29.0 (± 12.7, P<0.0011, Tukeys, n=4) and amplitude to a mean value of 43.9 (± 11.3, P<0.0016, Tukeys, n=4). Ca changes paralleled these changes. Similarly, cholesterol was associated with a significant decrease in agonist-stimulated contractions. Significant decline in total force fell to a mean value of 28.7 (± 13.9, P<0.0036, Tukeys, n=3) and duration to a mean value of 40.1 (± 10.1, P30) (19%, odds ratio 1.5, 1.22-1.85 95% CI), frequently due to inadequate uterine contractions (5.7%, odds ratio 3.54, 2.17-5.78 95% confidence interval), compared with normal women. Thus Ca signalling, which is central to myometrial contractions is significantly influenced by cholesterol. The deleteriously effect of cholesterol loading on membrane microdomains may contribute to uterine quiescence and labour complications in obese women.