A novel method for studying the mobilization of free arachidonic acid (AA) in isolated intestinal epithelial cells is described. The method is based on labeling the cellular phospholipids with 14C-AA and studying the release of this 14C-AA on subsequent phospholipase activation. Cells of high viability were isolated from the small intestine of guinea pigs and incubated with 14C-AA for 2 h; most of the incorporated 14C-AA was then esterified into phosphatidylethanolamine and phosphatidylcholine. When the labeled cells were stimulated with the calcium ionophore A23187 in the presence of external calcium, they released significant amounts of AA. In contrast, the cells released no AA when stimulated with A23187 in the absence of external calcium or in the presence of chlorpromazine or 4-bromophenacyl bromide, both of which are known to inhibit phospholipase A2 activity. On the other hand, the cells released significant AA in response to exogenous phospholipase C from Clostridium perfringens. These findings indicate that AA release in intestinal cells may be caused by calcium-mediated phospholipase A2 activation or by products of microbial phospholipase C activity. They also suggest the further use of 14C-AA-labeled cells for studying agents and mechanisms that may influence the release of AA in the gastrointestinal tract.