In the presence of Li+, glucose, 2-ketoisocaproate and carbamylcholine induced the rapid formation of 3H-inositol phosphates in rat pancreatic islets prelabelled with 3H-inositol. The production of labelled inositol phosphates continued up to 20 min of incubation. Glibenclamide and ionophore A23187 had no significant effect on labelled inositol phosphate production. The effects of carbamylcholine and to a lesser extent, glucose were found to persist in the absence of added Ca2+, but both were strongly inhibited by excess EGTA. In general, the rise in 3H-inositol phosphate production was associated with a fall in lipid bound radioactivity, although the latter was found to occur more slowly, and was of a smaller magnitude than labelled inositol phosphate formation. The results suggest that nutrient secretagogues and cholinergic agonists stimulate hydrolysis of phosphoinositides in pancreatic islets by a phospholipase C mechanism. This effect is Ca2+-dependent, but probably not triggered by increased Ca2+ uptake into the islet.