The concurrent effect of indomethacin or aspirin on prostaglandins (PGs) biosynthesis and on cellular fatty acid efflux were compared. Studies with rabbit kidney medulla slices and with isolated perfused rabbit kidney showed a marked difference between the two non-steroidal anti-inflammatory drugs, with regard to their effects on fatty acid efflux from kidney tissue. While aspirin effect was limited to inhibition of PGs biosynthesis, indomethacin also reduced the release of free fatty acids. In medullary slices, indomethacin inhibited the Ca2+ stimulation of phospholipase A2 activity and the resulting release of arachidonic and linoleic fatty acids. In the isolated perfused rabbit kidney, indomethacin inhibited the basal efflux of all fatty acids as well as the angiotensin II — induced selective release of arachidonate. Indomethacin also blunted the angiotensin II — induced temporal changes in the efflux of all other fatty acids. Neither indomethacin nor aspirin affected significantly the uptake and incorporation of exogenous (14C)-arachidonic acid into kidney total lipid fraction. Our tentative conclusion is that indomethacin inhibits basal as well as Ca2+ or hormone stimulated activity of kidney lipolytic enzymes. This action of indomethacin reduces the pool size of free arachidonate available for conversion to oxygenated products (both prostaglandin and non-prostaglandin types). The non-steroidal anti-inflammatory drugs can therefore be divided into two groups: a) aspirin-type compounds which inhibit PGs formation only by interacting with the prostaglandin endoperoxide synthetase and b) indomethacin-type compounds which inhibit PG generation by both reduction in the amount of available arachidonate and direct interaction with the enzyme.