In cultured pancreatic islets from neonatal rats labelled with [3H] arachidonic acid, glucose stimulation prompted a fall in the labelled arachidonate concentration of phosphatidylinositol and a concomitant rise in 1,2 diacylglycerol and phosphatidic acid. The time course of glucose stimulation indicated that this early event was followed by an increased liberation of arachidonic acid and incorporation into arachidonate metabolites. Incubation of homogenates of glucose stimulated islets with both phosphatidylinositol and phosphatidylcholine specifically labelled with arachidonate in the 2-position acyl chain generated arachidonic acid. This indicated both phospholipase C with 1,2 diacylglycerol lipase and phospholipase A2 activities in the action of glucose. Calcium dependent arachidonic acid release was also seen from arachidonic acid labelled phosphatidic acid. The findings suggest multiple sources of islet arachidonic acid following glucose stimulation including phospholipase A2 hydrolysis of phosphatidic acid.