Muscle cells from human gallbladders (GB) with cholesterol stones (ChS) exhibit a defective contraction, excess cholesterol (Ch) in the plasma membrane and lower binding of CCK-1 receptors. These abnormalities improved after muscle cells were incubated with Ch-free liposomes that remove the excess Ch from the plasma membrane. The present studies were designed to investigate the role of caveolin-3 proteins (CAV-3) in the pathogenesis of these abnormalities. Muscle cells from GB with ChS exhibit a higher Ch levels in the plasma membrane that were mostly localized in caveolae and associated with parallel increases in the expression of CAV-3 in the caveolae when compared to those of GB's with pigment stones (PS). The overall number of CCK-1 receptors in the plasma membrane was not different between muscle cells from GB's with ChS and PS but they were increased in the caveolae in muscle cells from GB's with ChS. Treatment of muscle cells from GB's with ChS with a G_i3 protein fragment increased the total binding of CCK-1 receptors (from 8.3% to 11.2%) and muscle contraction induced by CCK-8 (from 11.2% to 17.3% shortening). However, G_q/11 protein fragment had no such effect. Moreover, neither fragment had any effect on muscle cells from GB's with PS. We conclude that the defective contraction of muscle cells with excessive Ch levels in the plasma membrane is due to an increased expression of CAV-3 that results in the sequestration of CCK-1 receptors in the caveolae probably by inhibiting the functions of G_i3 proteins.