In response to ATP, astrocytes accumulate inositol phosphates and release arachidonic acid (AA) from phospholipid stores, events which may be linked through intracellular calcium mobilization and activation of phospholipase A2. When cells were exposed to ATP in the presence of serotonin there was a dose-dependent inhibition of AA release, an effect which was reversed by methysergide. However, the accumulation of inositol phosphates due to ATP was elevated in the presence of serotonin, and this effect was again reversed by methysergide. The mechanism by which serotonin inhibits ATP-induced arachidonate mobilization does not, therefore, involve the purinergic receptor or its coupling to inositol phospholipid pools. The hydrolysis of polyphosphoinositides by serotonin and subsequent generation of inositol trisphosphate may deplete a specific intracellular calcium store normally available to ATP, the mobilization of which stimulates phospholipase A2 and thus AA release.