1. Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. 2. Primary astrocytes were cultured under standard conditions and prelabeled with [(14)C]DHA or with [(3)H]AA. Adenosine 5'-triphosphate (ATP) (20 micro M applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca(2+)-independent phospholipase A(2) (iPLA(2)), since it is blocked by the selective iPLA(2) inhibitor 4-bromoenol lactone (BEL, 5 micro M) and is not affected either by removal of Ca(2+) from extracellular medium or by suppression of intracellular Ca(2+) release through PLC inhibitor (U73122, 5 micro M). 3. AA release, on the other hand, which is stimulated by ATP, is attributed to Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)). AA release is abolished by U73122 and, by removal of extracellular Ca(2+), is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 micro M), a general inhibitor of intracellular PLA(2) s. 4. Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA(2) and 40 kDa protein reactive to iPLA(2) antibodies. 5. The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 micro M), and by the protein kinase A (PKA) activator dibutyryl-cAMP (1 mM). In contrast, release of AA is not affected by forskolin or dibutyryl-cAMP, but is almost completely blocked by 2,3-dideoxyadenosine (20 micro M) and inhibited by 34% by H89 (10 micro M), inhibitors of adenylyl cyclase and PKA, respectively. 6. Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. 7. Different sensitivities of iPLA(2) and cPLA(2) to Ca(2+) and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.