Cholesterol affects the ligand binding function of the oxytocin receptor in a highly specific manner. While the structurally-related cholecystokinin receptor shows a strong correlation between the membrane fluidity and its binding function, the oxytocin receptor behaves differently. A stringent and unique requirement of the affinity state of the oxytocin receptor for structural features of the sterol molecule has been found. The molecular requirements differ both from those postulated for sterol-phospholipid interactions and from those known to be necessary for the activity of other proteins. Employing a new detergent-free subcellular fractionation protocol, a two-fold enrichment of the oxytocin receptors (10-15% of total receptors) has been detected in the cholesterol-rich, caveolin-containing membrane domains of the plasma membrane. While most of the properties of the oxytocin receptors were indistinguishable in cholesterol-poor versus cholesterol-rich membrane compartments, high-affinity oxytocin receptors localised in caveolin-enriched low-density membranes showed about a 3-fold higher stability against thermal denaturation at 37 degrees C compared with the oxytocin receptors localised in high-density membranes. Moreover, addition of cholesterol to the cholesterol-poor high-density membranes fully protected the oxytocin receptors against thermal denaturation and partially rescued high-affinity oxytocin binding. Although the membrane fluidity of the caveolin-enriched domains was lower than that in the high-density membranes, there was no correlation between the stability of oxytocin receptors and the fluidity level of the membrane domains. Finally, in a molecular modelling approach a putative cholesterol binding motif on the extracellular surface of the oxytocin receptor was found.