It has been shown that p38 mitogen-activated protein (MAP) kinase is absolutely necessary for the cardioprotection of early ischemic preconditioning in the heart. Reorganization of actin cytoskeleton after translocation of HSP27, which is mediated by p38 MAP kinase, was reported to be necessary for the cardioprotective effect of early ischemic preconditioning. Although Rho and Rho kinase are reported to regulate reorganaization of actin filaments, it is unknown whether Rho–Rho kinase pathway is involved in the cardioprotective effect of early ischemic preconditioning. The aim of the present study is to determine the involvement of Rho–Rho kinase pathway in the protective effect of early ischemic preconditioning in the rat hearts. Dominant-negative Rho significantly reduced the hypoxia-reoxygenation-induced activation of p38 MAP kinase, and constitutive active Rho activated p38 MAP kinase in rat myoblast H9c2 cells. Y-27632, a specific Rho kinase inhibitor, concentration-dependently attenuated the post-ischemic recovery of left ventricular developed pressure by early ischemic preconditioning. Thus, Rho–Rho kinase pathway is, at least in part, involved in the mechanism of early ischemic preconditioning.