A fluoroimmunoassay (FIA) for 14,15-epoxyeicosatrienoic acid (14,15-EET) and 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), cytochrome P450 epoxygenase products of arachidonic acid, was developed using fluorescence polarization. 14-15-EET was hydrolyzed and analyzed as 14,15-DHET. 14,15-DHET was conjugated to thyroglobulin and a specific antibody was raised in rabbits. Both [3H8]14,15-DHET in radioimmunoassay or fluorescein-labeled 14,15-DHET (14, 15-DHET*) in FIA bound to this antibody and were competitively displaced by 14,15-DHET. The binding activity and cross-reactivity of 14,15-DHET antibody were also studied by RIA compared to FIA. The antibody cross-reacted < or = 1% with 11,12-DHET and 14,15-EET and < 0.1% with other regioisomeric DHETs and arachidonic acid metabolites. The detection limit of 14,15-DHET was 2 pg/0.6 ml by FIA. Using this method, we found that A23187 stimulated the production of 14,15-EET by endothelial cells by angiotensin II stimulated 14,15-EET release from zona glomerulosa cells. The production of 14,15-EET in these samples was confirmed by gas chromatography/mass spectrometry. These studies demonstrate a sensitive and specific FIA for 14,15-EET and 14,15-DHET and that agonists stimulate the release of these eicosanoids in two cell types, bovine coronary artery endothelial cells and bovine zona glomerulosa cells.