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The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydr oxyeicosatrienoic acid (5,6-DHT) to 5,6-δ-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-δ-lactone and 5,6-DHT with a t1/2 ≈ 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (λ = 205) comigrating with 5,6-δ-lactone. Incubation of 5,6-δ-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 ± 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 ± 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 ± 5.5 pg/ml and 21.6 ± 6.3 pg/ml, respectively, by arachidonic acid.—Fulton, D., J. R. Falck, J. C. McGiff, M. A. Carroll, and J. Quilley. A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol.