To determine the mechanisms of Ca2+ mobilization induced by receptor agonists, we examined the role of Rho-kinase on the sarcoplasmic reticulum (SR) Ca2+ stores-dependent and -independent Ca2+ influx in guinea pig tracheal smooth muscle (TSM). Isometric tension and intracellular Ca2+ concentration ([Ca2+]i) were simultaneously measured using fura-2-loaded tissues. Depletion of the SR Ca2+ stores by thapsigargin caused an increase in [Ca2+]i and contraction, demonstrating capacitative Ca2+ entry (CCE). Because CCE was not inhibited by nifedipine, voltage-operated Ca2+ channels are not involved in CCE. Under the condition that CCE is fully activated, methacholine (MCh) and histamine caused further increases in [Ca2+]i and tension, demonstrating noncapacitative receptor-operated Ca2+ entry (non-CCE). The Ca2+ influx and contraction via non-CCE was inhibited by Y-27632, a Rho-kinase inhibitor, in a concentration-dependent fashion. In contrast, Y-27632 did not affect thapsigargin-induced CCE. Cytochalasin D, which disrupts actin cytoskeleton, inhibited contraction induced by CCE or MCh with no change in [Ca2+]i. Our results indicate that not only CCE but also non-CCE exist in TSM and that the latter is regulated by Rho-kinase, independent of actin cytoskeleton. In conclusion, Ca2+ influx regulated by the RhoA/Rho-kinase pathway may play a functional role in contraction by agonists.