The consequences of macrophage membrane incorporation of (n-3) polyunsaturated fatty acids (PUFA) on diradylglycerol (DG) formation and phospholipase-mediated signal transduction in vivo remain largely unknown. Three-to four-week-old C57BL/6 mice were randomly assigned to diets in which the 10% lipid component of the purified diet was either corn oil ethyl ester (control) or (n-3) ethyl ester [(n-3) PUFA-enriched]. After 4 wk, thioglycollate-elicited peritoneal macrophages were harvested and assayed for (1) total DG mass, (2) DG molecular species fraction analyses by argentation TLC and (3) separation of phospholipid classes and analyses of their molecular species fractions. We found that basal DG mass and the increase in DG mass after stimulation with platelet-activating factor or phorbol myristate acetate were similar in cells from control and (n-3) PUFA-enriched diet groups, whereas ionomycin-stimulated DG formation was less in the cells from the (n-3) PUFA-enriched diet group. Analyses of DG molecular species fractions showed that the proportions of species with five and six double bonds were markedly greater in the (n-3) PUFA-enriched diet group macrophages compared with those of the control diet group. The molecular species fractions of all phospholipid classes including phosphatidylinositol showed substantial incorporation of (n-3) PUFA. These results demonstrate that dietary enrichment with (n-3) PUFA induces marked changes in DG metabolism in murine peritoneal macrophages. These changes may contribute to the mechanisms of antiinflammatory, antithrombotic and antineoplastic actions associated with dietary (n-3) PUFA supplementation.