Introduction and objective: Caveolae and lipid rafts are membrane microdomains that are known to concentrate components of the G_s signalling pathway. Cyclic AMP (cAMP) is generated following G _s activation which induces vascular smooth muscle relaxation and thereby affecting blood pressure. The aim of this study was to examine how disruption of caveolae and lipid rafts affects vascular smooth muscle contractility with special reference to the G _s pathway. Methods: Tail artery preparations from mice, rats, and caveolin-1- deficient mice were mounted for force registration and stimulated with various G protein-coupled receptor (GPCR) agonists. Caveolae/lipid rafts were pharmacologically disrupted by cholesterol depletion using methyl- b-cyclodextrin (mbcd). Western blotting using an antibody detecting protein motifs phosphorylated by protein kinase A (PKA) was exploited as a read-out of G_s activation. Results: Pharmacological disruption of lipid rafts/caveolae converts the GPCR agonist Arg⁸-vasopressin from a vasoconstrictor to a vasodilator. The increased relaxation observed in cholesterol depleted arteries was inhibited by the cAMP antagonist, Rp-8-Br-cAMPs, but unaffected by the nitric oxide synthase inhibitor L-NAME. Vasopressin- induced contraction was also impaired in arteries from caveolin-1-deficient mice. Cholesterol depletion and caveolin-1-gene ablation were non-additive. Phosphorylation of several putative PKA substrates increased following cholesterol depletion. Conclusions: The disruption of caveolae favours G_s -coupling of some receptors that traditionally are considered to couple to G_q and suggests that promiscuity in receptor-coupling is avoided by compartmentalisation of the G_s pathway in caveolae in the vasculature. The favourable conditions for G_s- activation following cholesterol depletion could explain impaired contractility for several GPCRs.