Uterine quiescence is essential for successful pregnancy. Cholesterol and triglycerides are markedly increased in pregnancy. Cholesterol is enriched in micro domains of the plasma membrane known as rafts and caveolae. Both lipid rafts and caveolae have been implicated in cellular signalling cascades. The purpose of this work was to investigate if manipulation of cholesterol content alters uterine contractility. Late pregnant (19-21 days) rats were humanely killed and then strips of longitudinal myometrium dissected. Force and Ca²⁺ measurements were simultaneously recorded and cholesterol increased, by addition of cholesterol (5mg/ml) or low density lipoproteins (LDLs) (0.25mg/ml), or reduced by methyl--cyclodextrin (2% MCD) or cholesterol oxidase (2U/ml), addition to the perfusate. Both LDL's and cholesterol profoundly inhibited spontaneous uterine force production and associated Ca²⁺ transients; frequency, amplitude and duration of contraction were all significantly reduced compared to preceding control contractions. Force and Ca²⁺ were also reduced by cholesterol when oxytocin (1nM) was used to stimulate the myometrium. Uterine activity was significantly increased by cholesterol extraction with MCD or cholesterol oxidase treatment. Electronmicroscopy (EM) confirmed the lipid raft disrupting effect of MCD, as formerly EM-visible caveolae in the myometrial cell membrane all but disappeared following MCD treatment. These data show that uterine smooth muscle cell cholesterol content is critically important for functional activity. A novel finding of our study is that cholesterol is inhibitory for force generation. It may be one of the mechanisms operating to maintain uterine quiescence throughout gestation and may also contribute to difficulties in labour suffered by obese women.