The effects of Ca2+ and acetylcholine on the degradation and synthesis of phosphatidylinositol in rabbit vas deferens was studied in vitro by a pulse--chase technique and by measuring the content of the phospholipid in the tissue. Ca2+-dependent degradation of phosphatidylinositol was found in slices and homogenates prelabelled with myo-[2-3H]inositol. The phosphatidylinositol content of the slices also decreased by a Ca2+-dependent mechanism. On the other hand, removal of intracellular Ca2+ with the ionophore A23187 and EGTA increased the amount of phosphatidylinositol. These results indicate that the intracellular Ca2+ concentration has an important role in regulating the phosphatidylinositol content of the tissue. Increasing the extracellular K+ concentration, which causes an increase in plasma-membrane Ca2+ permeability, did not enhance phosphatidylinositol breakdown nor decrease its tissue content. However, phosphatidylinositol synthesis was clearly inhibited. After stimulation of the smooth muscle with acetylcholine, degradation of phosphatidylinositol was enhanced. Furthermore, the content of phosphatidylinositol in the tissue also decreased. These phenomena were evident even in the absence of Ca2+. The acetylcholine-induced degradation of phosphatidylinositol was blocked by the muscarinic antagonist atropine, but not by the nicotinic antagonist (+)-tubocurarine. The acetylcholine-induced decrease in the phosphatidylinositol content of the tissue led to the compensatory synthesis of phosphatidylinositol. Synthesis was separated from degradation in the same tissue. Compensatory synthesis was inhibited by acetylcholine. The degradation of phosphatidylinositol induced by acetylcholine was not inhibited by 8-bromoguanosine 3':5'-cyclic monophosphate, indicating that the degradative process was not mediated by an increase in the cyclic nucleotide.