The role of a GTP-binding protein in activation of phospholipase C in bovine corneal epithelium was determined by investigating the effects of non-hydrolyzable GTP analog, GTP-γ-S, and NaF on breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) in this tissue. GTP-γ-S (2–50μM), when introduced into the permeabilized corneal epithelial cells labeled with myo-[3H]inositol, dose-dependently increased the formation of myoinositol trisphosphate (IP3). Other guanine nucleotides and ATP were ineffective. Incubation of32P-prelabeled corneal epithelium with NaF (2–50 mM) resulted in increased breakdown of PIP2and increased synthesis of phosphatidic acid. In myo-[3H]inositol-labeled tissue, NaF dose-dependently increased the accumulation of IP3. Microsomal membrane fraction from corneal epithelium was found to contain phospholipase C activity towards endogenous phosphatidylinositol 4-phosphate and PIP2. The enzyme activity was stimulated by Ca2+(100μM). Addition of GTP-γ-S to microsomal fraction containing phosphoinosi-tides which were radiolabeled with32Piin situor with [γ-32P]ATPin vitrocaused a dose dependent hydrolysis of PIP2. These data, taken collectively, suggest that a GTP-binding protein is involved in activation of phospholipase C towards PIP2in bovine corneal epithelium, and that this guanine nucleotide regulatory protein may serve to couple norepinephrine and 5-hydroxytryptamine receptors to phospholipase C during transmembrane signalling.