To define the mechanisms by which fish oil protects rats with passive Heymann nephritis (PHN) from proteinuria in vivo, we investigated whether omega-3 fatty acid substitution of glomerular epithelial cells (GEC) in culture alters their susceptibility or response to complement-mediated sublethal injury. The results show that GECs can be cultured under conditions that effectively incorporate omega-3 or omega-6 fatty acids into membrane phospholipids without causing toxicity. Under these conditions, sublethal injury with anti-Fx1A and C5b-9 stimulated a 6.6-fold increase in TxA2 production by GECs substituted with arachidonic acid (AA, omega-6) but no increase was detected in eicosapentaenoic acid (EPA, omega-3) substituted cells. Sublethal cell membrane injury was of equal severity in both groups as measured by the release of preloaded biscarboxyethyl carboxyfluorescein and by the transepithelial flux of albumin. In addition, omega-3 and omega-6 fatty acid substituted cells showed similar increases in diacylglycerol mass in response to sublethal injury by C5b-9, suggesting that omega-3 incorporation did not limit phospholipid (PL) hydrolysis by PLC. From this we can conclude that the protective effect of fish oil in PHN does not appear to result from the preservation of GEC integrity but is likely related to changes in the production of lipid mediators.