A translocation of protein kinase C (PKC) from cytosol to plasma membrane has been reported as an association with agonist-induced Ca²⁺ sensitization in smooth muscle contraction. Therefore, it is possible that a downstream target of PKC, CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa), might also be translocated to membrane when activated. To confirm this hypothesis, cytosolic and membrane CPI-17 was measured in acetylcholine (ACh)- and high K⁺ depolarization-stimulated bronchial smooth muscle of rats. An active form of CPI-17, i.e., Thr38-phosphorylated CPI-17, was also measured in cytosolic and membrane fractions. Immunoblot analyses demonstrated a translocation of CPI-17 from cytosolic to membrane fraction by ACh-, but not high K⁺ depolarization-, stimulation in time- and concentration-dependent manners. Interestingly, phosphorylated CPI-17 was detected only in membrane fractions in the ACh-stimulated tissues. However, in the high K⁺ depolarization-stimulated tissues, phosphorylated CPI-17 was not detected both in membrane and cytosolic fraction. To estimate downstream of activated-CPI-17, immunoblotting for phosphorylated myosin light chain (MLC) was performed in ACh- or high K⁺ depolarization-stimulated tissues. ACh- and high K⁺ depolarization-induced phosphorylation of MLC was observed in its contraction-dependent manner. In conclusion, we for the first time suggested that CPI-17 is translocated and phosphorylated by ACh, but not high K⁺ depolarization, in rat bronchial smooth muscle. ACh-induced translocation and phosphorylation of CPI-17 might be caused via the activation of muscarinic receptor.