The involvement of phosphatidylcholine (PC) hydrolysis in the regulation of insulin secretion was studied in mouse pancreatic islets prelabelled with [3H]choline. Phospholipase C (PLC) and phospholipase D (PLD) activities were demonstrated and also that of an enzyme that removes both fatty acids from PC and thus catalyses the production of [3H]glycerophosphorylcholine (GroPCho). After 2 min of incubation with 20 mm glucose a 35% increase in the content of [3H]GroPCho was observed in prelabelled islets, whereas the amount of [3H]lysoPC, [3H]phosphorylcholine (PCho) and [3H]choline was unaffected. After 30 min of incubation with 20 mm glucose, 0·2 mm tolbutamide, 40 mm KC1, 10 mm succinic acid monomethyl ester (SME) or 10 mm NaF, a 25-50% increase in [3H]GroPCho was observed. In the presence of 100 μm diazoxide or 35 μm RHC 80267 the glucose activation was attenuated. PLC was stimulated slightly by tolbutamide and 100 μm isoprenaline (isoproterenol), whereas SME decreased the amount of [3H]PCho by 10%. [3H]Choline content was increased by 25-40% in the presence of 0·16 μm 12-O-tetradecanoylphorbol 13-acetate (TPA), 10 mm NaF or 100 μm carbachol. This effect of fluoride was potentiated in the presence of 20 mm glucose. It is concluded that metabolism of PC to GroPCho may be involved in the regulation of glucose-stimulated insulin secretion, and that PLD may participate in insulin secretion evoked by TPA, carbachol and fluoride.