Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.