After in vitro incubation of mouse pancreas for 80 min, the levels of diacylglycerol (expressed in μmol/g wet weight) in unstimulated tissue and in tissue incubated with 10 μM or 100 μM acetylcholine (plus 100 μM eserine) were, respectively, 0.51, 1.30 and 3.73. Increases in palmitic, oleic and linoleic acids in the diacylglycerol pool accounted for 75% of the total increase. With 100 μM acetylcholine, the proportions of individual fatty acids in diacylglycerol showed a significant enrichment of stearic and arachidonic acid at 40 min and of linoleic and arachidonic acids at 80 min. With the exception of an enrichment of stearic and arachidonic acids, the fatty acid composition of the new diacylglycerol resembled that of the triacylglycerol pool more closely than that of other classes of lipid in the pancreas.