In both the in vivo and in vitro experiments, the changes in levels of stearic and arachidonic acids in diacylglycerol indicated that only a small proportion of the total increase in diacylglycerol level could have been derived from the breakdown of stearoyl, arachidonoyl phosphatidylinositol which occurs in response to cholinergic stimulation in the pancreas. The major effect of acetylcholine in diacylglycerol metabolism in mouse pancreas is separate, therefore, from the effect on phosphatidylinositol metabolism.