Lipids, including cholesterol, are critical components of the cell membrane where they are enriched in microdomains, lipid rafts, which organize and concentrate receptors and intracellular proteins involved in signal transduction. The present study examined the effects of cholesterol depletion on serotonin (5-HT) binding and signaling via 5-hydroxytryptamine(7) (5-HT(7)) receptors in stably transfected HeLa cells. Immunohistochemical, ligand-binding and biotinylation experiments demonstrated that the studied cells expressed high levels of 5-HT(7) receptors at their surface with a pharmacological profile resembling 5-HT(7) receptors in native tissue. Depletion of cholesterol, by combined treatment with mevastatin, fumonisin B(1) and mevalonate or methyl-beta-cyclodextrin (MbetaCD), caused highly significant reductions in the B(max) values of [(3)H]5-HT- and [(3)H]-(R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol ([(3)H]SB269970)-binding to 5-HT(7) receptors. Cholesterol depletion also reduced the total level of 5-HT(7) receptor protein determined by Western blot analysis. None of the examined treatments affected the affinity of [(3)H]5-HT- or [(3)H]SB269970-binding to 5-HT(7) receptors. Treatment with serotonin caused strong inductions in the phosphorylation states of Ser(63)-ATF-1 and Ser(133)-CREB. These effects of serotonin on signal transduction were significantly counteracted by pre-treatment with cholesterol synthesis inhibitors. Altogether, the present study demonstrates that cholesterol depletion decreases binding of both agonist and antagonist radioligands to 5-HT(7) receptors and counteract 5-HT(7) receptor-mediated intracellular signaling.