A method for studying the mobilization of free arachidonic acid (AA) in viable isolated human intestinal epithelial cells has been developed and applied to the study of patients with Crohn's disease. Cells were isolated from morphologically unaffected parts of the distal ileum and incubated with 14C-AA; most of the incorporated 14C-AA was then found in phospholipids (mainly phosphatidylcholine) and in a pool of neutral lipids (mainly triacylglycerols). Cells from patients with Crohn's disease incorporated more 14C-AA into their neutral lipids than did cells from control patients. When the labeled cells were stimulated with phospholipase C from Clostridium perfringens or with the calcium ionophore A23187, they released significant amounts of AA, mainly from phosphatidylcholine. There was no difference between cells from Crohn patients and controls in the 14C-AA amounts released, but unstimulated and phospholipase C-stimulated cells from prednisolone-treated Crohn patients released less AA than cells from control patients. The A23187-stimulated AA release was completely inhibited by the phospholipase A2 inhibitor 4-bromophenacyl bromide, whereas the phospholipase C-stimulated release was not. These findings suggest that AA release in human small-intestinal epithelial cells may be caused by calcium-mediated phospholipase A2 activation or by products of microbial phospholipase C activity and that prednisolone reduces the mobilization of free AA in intestinal epithelial cells. They also illustrate the potential use of isolated epithelial cells for revealing mechanisms underlying AA release in the intestinal mucosa in different disease states.