1. The turnover of phosphatidylinositols and other glycerolipids was examined in rat pancreatic fragments incubated in the presence of carbamylcholine and pancreozymin used at a concentration inducing maximal alpha-amylase hypersecretion. 2. In stimulated tissue, [1-14C]acetate-labeled fatty acids were incorporated into phosphatidylinositols, 1,2-diacylglycerols, and phosphatidic acids in preference to phosphatidylcholines, phosphatidylethanolamines, triacylglycerols, monoacylglycerols, and free fatty acids. Variations in the percent distribution of 14C among fatty acids and in specific activity of individual fatty acids in each lipid class suggested that the secretagogues reduced selection of newly synthesized 1,2-diacylglycerols which occurred in the resting state before their incorporation into phosphatidylinositols. Secretagogues also promoted recycling of endogenous 1,2-diacylglycerols (produced from hydrolysis of unlabeled glycerolipids) for the biosynthesis of phosphatidylinositols. 3. Increased rate of incorporation of [1-14C]palmitate, [1-14C]linoleate, [1-14C]arachidonate and [1(3)(n)-3H]glycerol into phosphatidylinositols was detrimental to phosphatidylcholines. 4. The lipolytic effects of carbamylcholine and pancreozymin as illustrated by the release of 1,2-diacylglycerols and free fatty acids, were markedly inhibited in calcium-free medium enriched with 1 mM EGTA but increased turnover of phosphatidylinositols as determined from incorporation of radioactive precursors was only moderately affected.