Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H1) and 5-hydroxytryptamine (serotonin) receptors (5-HT2A) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-β-cyclodextrin (MβCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (∼80%), 5-HT (100%), α-methyl-5-HT (100%), and KCl (∼30%). Cholesterol-loaded MβCD (CL-MβCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (∼85%) and 5-HT (∼59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MβCD and partially reestablished by CL-MβCD. The increase in intracellular Ca2+ that was induced by both agonists was reduced by MβCD. The presence of caveolin-1 (Cav-1), H1, 5-HT2A, and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H1 receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT2A receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H1 and 5-HT2A receptors through their Rho/ROCK signaling.