The main membrane proteins of caveolae (caveolin-1, −2 and −3) oligomerize within lipid rich domains of smooth muscle plasma membrane and participate in the regulation of G protein-coupled receptor signaling. AIM To identify the role of caveolin-1 in muscarinic m3-receptor mediated stimulation of Rho kinase and ZIP kinase activity in gastric smooth muscle. METHODS Muscle cells were isolated from rabbit stomach, and from wild type and caveolin-1−/− mice. Carbachol (CCh)-induced stimulation of Rho kinase and ZIP kinase activity was determined by immunokinase assay. Muscle contraction was measured by scanning micrometry. RESULTS CCh (0.1 μM) caused stimulation of both Rho kinase and ZIP kinase activity in gastric muscle cells. Y27632 (1 μM), a selective blocker of Rho kinase activity inhibited stimulation of both Rho kinase and ZIP kinase activity suggesting that Rho kinase is an upstream kinase of ZIP kinase. Disruption of caveolae with methyl β-cyclodextrin or suppression of caveolin-1 with caveolin-1 siRNA attenuated the increase in Rho kinase and ZIP kinase activity in response to CCh. Similarly, Rho kinase activation and sustained contraction in response to CCh were significantly attenuated in muscle cells isolated from caveolin-1−/− mice compared to wild type mice. CONCLUSION In gastric smooth muscle caveolin-1 positively regulates Rho kinase/ZIP kinase-dependent sustained muscle contraction.