Work from our laboratory has previously demonstrated the requirement of membrane cholesterol in the function of the serotonin(1A) receptor, a member of the G-protein Coupled receptor (GPCR) superfamily. In order to monitor the effect of cellular organization on the function of human serotonin(1A) receptors, we explored receptor function following cholesterol depletion in live cells and membranes isolated from cholesterol-depleted cells. We report here the novel observation that while ligand binding of serotonin(1A) receptors displays an increase in membranes isolated from cholesterol-depleted cells, such trend is absent when binding is performed on cholesterol-depleted intact cells. Importantly, we show here, for the first time, that G-protein coupling of the serotonin(1A) receptor is enhanced in membranes isolated from cholesterol-depleted cells. These results assume pharmacological relevance in view of the recently described structural evidence of specific cholesterol binding sites in GPCRs, and may help in designing better therapeutic strategies for diseases related to GPCRs. (C) 2009 Elsevier Inc. All rights reserved.