The chemokine receptor CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains. Here we elucidate the effects of membrane fluidity on CCR5 signalling and expression using the monocytic THP-1 cells. MCD treatment of THP-1 cells, which removes nearly all cholesterol from the plasma membrane, leads to an increase in the signalling properties of CCR5. In contrast, the prevention of cholesterol production with lovastatin and simvastatin decreases the release of intracellular calcium and also decreases receptor cell surface expression. The loss of response in lovastatin treated cells can be rescued by MCD addition, which shows that the cholesterol content in the membrane is only one factor in determining the amount of receptor response. We show that CCR5 signalling is dependent on thapsigargin-sensitive Ca2+ stores and on activation of ryanodine receptors as well as InsP3 receptors or store-operated channels. Cholesterol depletion with MCD changes the thapsigargin sensitivity in THP-1 cells and also alters receptor-G-protein coupling towards pertussis toxin (PTX) independent G-proteins. Cholesterol removal by MCD in THP-1 cells has far reaching consequences for receptor activation and signalling and emphasises the need for a clearer understanding of how membrane fluidity affects receptor signalling events.