It is widely recognized that in addition to regulating the expression and activity of arachidonic acid (AA)-metabolizing enzymes, the availability of free AA limits eicosanoid biosynthesis. AA participates in a deacylation/reacylation cycle of membrane phospholipids in which the fatty acid is cleaved by phospholipase A2 and reesterified by acyltransferase. Thus, free AA can become available either by phospholipase A2 activation or by inhibition of fatty acid reincorporation. We observed that exposure of [3H]AA-prelabeled macrophages to micromolar concentrations of unlabeled AA resulted in a net release of 3H-radioactivity into the extracellular medium. This was not the consequence of phospholipase A2 activation, but of impaired reesterification of previously liberated [3H]AA. The eicosanoid precursors eicosatrienoic acid (ETA) and eicosapentaenoic acid (EPA) mimicked the effect of unlabeled AA on 3H-radioactivity release from [3H]AA-prelabeled macrophages, but all other fatty acids tested were ineffective. Similarly, only AA, ETA and EPA were able to inhibit [3H]AA uptake by macrophages, all other fatty acids being ineffective. From these data, it is concluded that intact macrophages contain an acylating system specific for the eicosanoid precursors AA, ETA and EPA. Altogether, the results of this study underscore the importance of fatty acid reacylation in controlling free AA levels in macrophages.