Preincubation of rat pancreatic islets with 3H-inositol, and subsequent exposure, in the presence of LiCl, to either glucose or carbamylcholine resulted in a rapid stimulation of 3H-inositol 1,4,5-triphosphate and 3H-myo-inositol 1,4-bisphosphate formation, the level of which reached a plateau after about 5 min of stimulation. Both stimuli also caused an approximately linear accumulation of 3H-myo-inositol 1-phosphate. The amounts of 3H-inositol phosphates formed were dependent on the concentration of LiCl. Studies of 32P-labeling of islet ATP, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 4-phosphate revealed that these approached isotopic equilibrium after about 240-min incubation, whereas 32P-labeling of phosphatidylinositol, phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine proceeded at a lower rate. Carbamylcholine provoked an immediate fall in 32P-PtdIns(4,5)P2 and, to a lesser extent, 32P-phosphatidylinositol 4-phosphate. Glucose caused a similar response although, in this case, the most marked decline was in a more polar 32P-labeled lipid. Cholecystokinin-pancreozymin was also found to induce 32P-PtdIns(4,5)P2 hydrolysis, although the ionophore A23187 was without effect. Both carbamylcholine and glucose induced an increase in 32P-phosphatidic acid. The results provide two independent pieces of evidence suggesting that phospholipase C-mediated hydrolysis of polyphosphoinositides occurs as an early response in rat islets to either nutrient or neurotransmitter secretagogues.