The signal transduction that mediates CCK-induced contraction of gallbladder muscle was investigated in the cat. Contraction was measured by scanning micrometry in single muscle cells isolated enzymatically with collagenase. Production ofd-myo-inositol 1,4,5-trisphosphate (IP₃) andsn-1,2-diacylglycerol (DAG) was quantitated using HPLC and TLC, respectively. Protein kinase C (PKC) activity was determined by measuring the phosphorylation of a specific substrate peptide from myelin basic protein, Ac-MBP-(4—14). CCK-induced contraction was blocked by incubation in strontium medium, pertussis toxin (PTx), and antibodies against G_iα₃or βγ-subunits but was not blocked by Ca²⁺-free medium or by antibodies against G_q/11α, G_iα_1–2, or G_oα. The contraction induced by CCK was inhibited by the phospholipase C (PLC) inhibitor U-73122, anti-PLC-β3 antibody, and the IP₃ receptor antagonist heparin but was not inhibited by the the phospholipase D inhibitor propranolol or antibodies against PLC-β1 or PLC-β2. Western blot analysis of gallbladder muscle revealed the presence of PLC-β2 and PLC-β3 but not PLC-β1. CCK caused a 94% increase in IP₃ generation and an 86% increase in DAG generation. A low dose of CCK caused PKC translocation, and CCK-induced contraction was blocked by the PKC inhibitor H-7. A high dose of CCK, however, caused no PKC translocation, and its contraction was blocked by the calmodulin antagonist CGS9343B. In conclusion, CCK contracts cat gallbladder muscle by stimulating PTx-sensitive G_{i 3} protein coupled with PLC-β3, producing IP₃ and DAG. Low doses activate PKC, whereas high doses activate calmodulin.