The serotonin(IA) (5-HTIA) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We report here that solubilization of the hippocampal 5-HTIA receptor by the zwitterionic detergent CHAPS is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity and extent of G-protein coupling. Importantly, replenishment of cholesterol to solubilized membranes using MbetaCD-cholesterol complex restores the cholesterol content of the membrane and significantly enhances the specific agonist binding activity and G-protein coupling. These novel results provide useful information on the role of cholesterol in solubilization of G-protein-coupled receptors, an important step for molecular characterization of these receptors. (C) 2004 Elsevier Inc. All rights reserved.