Ca²⁺ sensitivity of arterial contractility is governed by regulating myosin phosphatase activity in response to agonist stimuli. CPI-17, a myosin phosphatase inhibitor phosphoprotein, is phosphorylated concomitantly with agonist-induced contractile Ca²⁺ sensitization in mammalian artery. CPI- 17 was not detected in chicken artery but readily detected in pigeon artery. To evaluate a role of CPI- 17, we compared contractility of the arteries of "CPI-17- deficient" chicken with those of CPI-17-rich rabbit and pigeon, and studied the effect of CPI-17-reconstitution in chicken artery. Other major regulatory/contractile proteins for Ca²⁺ sensitization are expressed in both chicken and rabbit arteries. Agonists, such as 1-agonist and endothelin-1, produced significant contraction in arteries of all species under physiological Ca²⁺-containing conditions. Depletion of Ca²⁺ abolished these contractions in chicken but partially inhibited them in rabbit and pigeon arteries. Unlike CPI-17-rich tissues, chicken arteries exerted little Ca²⁺ sensitization in response to 1-agonist or endothelin-1. GTPS slightly produced Ca²⁺ sensitizing effect in chicken artery, but significantly smaller compared with CPI-17-rich tissues. A PKC activator (PDBu) did not generate but rather reduced a contraction in both intact and - toxin-permeabilized chicken artery in contrast to a large contraction in CPI-17-rich arteries. Myosin light chain phosphorylation was reduced by PDBu in chicken but elevated in rabbit artery. Addition of recombinant CPI- 17 into -escin-permeabilized chicken artery restored PDBu-induced and enhanced GTPS-induced Ca²⁺ sensitization. Thus, CPI-17 is essential for G protein/PKC-mediated Ca²⁺ sensitization in smooth muscle.