To clarify whether cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) activation and Rho-kinase inhibition share a common mechanism to decrease the Ca²⁺ sensitivity of airway smooth muscle contraction, we examined the effects of 8-Bromo-cAMP (8-Br-cAMP), a stable cyclic AMP analogue, and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a Rho-kinase inhibitor, on carbachol (CCh)-, guanosine 5'-O-(3-thiotriphosphate) (GTPS)-, 4-phorbol 12, 13-dibutyrate (PDBu)-, and leukotriene D₄ (LTD₄)-induced Ca²⁺ sensitization in a-toxin-permeabilized rabbit tracheal and human bronchial smooth muscle. In rabbit trachea, CCh-induced smooth muscle contraction was inhibited by 8-Br-cAMP and Y-27632 to a similar extent. However, GTPS-induced smooth muscle contraction was resistant to 8-Br-cAMP. In the presence of a saturating concentration of Y-27632, PDBu-induced smooth muscle contraction was completely reversed by 8-Br-cAMP. Conversely, PDBu-induced smooth muscle contraction was resistant to Y-27632. In the presence of a saturating concentration of 8-Br-cAMP, GTPS-induced Ca²⁺ sensitization was also reversed by Y-27632. The 8-Br-cAMP had no effect on the ATP-triggered contraction of tracheal smooth muscle that had been treated with calyculin A in rigor solutions. The 8-Br-cAMP and Y-27632 additively accelerated the relaxation rate of PDBu- and GTPS-treated smooth muscle under myosin light chain kinase (MLCK)-inhibited conditions. In human bronchus, LTD₄^-induced smooth muscle contraction was inhibited by both 8-Br-cAMP and Y-27632. In conclusion, cAMP/PKA-induced Ca²⁺ desensitization contains at least two mechanisms: inhibition of the muscarinic receptor signaling upstream from Rho activation, cyclic AMP/PKA preferentially reverses PKC-mediated Ca²⁺ sensitization in airway smooth muscle.