In the present study, the effects of a selective Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor, Y-27632 [(+)-( R)- trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] on acetylcholine-induced contraction and Ca 2+ sensitization of rat bronchial smooth muscle were examined. Intact and β-escin-permeabilized muscles of the third branch of intrapulmonary bronchi were used. In intact muscles, Y-27632 (10 −6–10 −4 M) concentration-dependently inhibited acetylcholine-induced contractile responses. In acetylcholine (10 −3 M)-precontracted intact muscles, the maximal relaxation (about 50% inhibition of contraction) was obtained by a concentration of 10 −4 M Y-27632, which had no effect on the resting tone. In β-escin-permeabilized muscles, addition of acetylcholine (10 −5–10 −3 M) plus GTP (100 μM) induced a further contraction, i.e., Ca 2+ sensitization at a constant Ca 2+ concentration of pCa=6.0. The acetylcholine-induced Ca 2+ sensitization was completely blocked in the presence of 10 −4 M Y-27632, whereas the Ca 2+-induced contraction itself was not affected by Y-27632. Immunoblot study revealed the expression of ROCK-I and ROCK-II proteins in the intrapulmonary bronchi of rats. These findings suggest that Y-27632 dilates acetylcholine-mediated contraction of rat bronchial smooth muscle by inhibiting RhoA/ROCK-mediated Ca 2+ sensitization.