BACKGROUND. Gallbladder disease is prevalent during pregnancy. It has been suggested that this complication of pregnancy is due to increased bile Ch induced by estrogens and to gallbladder hypomotility caused by increasing levels of progesterone (P4). Studies on non-pregnant gallbladders have shown that increased levels of bile Ch contribute to both gallstone formation and bile stasis. AIM: these studies were designed to investigate the interactions between high levels of plasma membrane Ch and P4 on human and guinea pig gallbladder muscle. METHODS: Contraction studies were performed in intact and permeabilized gallbladder muscle cells. Muscle cells were transfected with CAV-3 siRNA, G proteins were determined by Western blot and ³H-P4 incorporation by muscle cells was measured in the -scintillation counter. RESULTS: High caveolar Ch blocked the effects induced by 6 h P4 treatment. High Ch levels suppressed the expected P4 inhibition of GTPS induced contraction as well as changes in G proteins, down regulation of Gi3 and up regulation of Gs proteins. Ch inhibited the P4 actions at the level of CAV-3 since its effects were antagonized by CAV-3 Ab and by reducing its expression through CAV-3 siRNA. CAV-3 Ab and siRNA reduced caveolar Ch levels. High Ch levels and CAV-3 Ab blocked the incorporation of ³H-P4 into caveolae. GDPS treatment had no effect on P4 actions. CONCLUSION: High caveolar Ch levels blocked the P4 effects on muscle contraction and G proteins changes probably because both Ch and P4 require CAV-3 proteins for their transport across the plasma membrane.