Renal glomerular and cortical metabolism of endogenous arachidonic acid by cytochrome P-450 epoxygenase yields 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EET). Using gas chromatography-mass spectrometry, we measured the synthesis of 8,9-EET from an endogenous pool of arachidonic acid in normal rat kidney. The (8S,9R) isomer was favored over the (8R,9S) isomer in a ratio (%) of 59 to 41 in isolated glomeruli and 68 to 32 in cortex tissue. (8S,9R)- but not (8R,9S)-EET elicited dose-dependent vasoconstriction on intrarenal administration in the euvolemic Munich-Wistar rat. Micropuncture measurements of glomerular dynamics revealed that (8S,9R)-EET increased afferent arteriolar resistance (RA) leading to reductions in single-nephron plasma flow rate (QA), net transcapillary hydraulic pressure difference (delta P), and consequently single-nephron glomerular filtration rate (SNGFR). There was no significant change in the value of the glomerular capillary ultrafiltration coefficient (Kf). In the presence of a cyclooxygenase inhibitor, indomethacin, the effects of 8,9-EET were reversed. RA fell leading to increases in QA and delta P, with resultant augmentation of SNGFR. Under these conditions, a modest reduction if Kf was noted. Thus (8S,9R)-EET is a stereoselective renal vasoconstrictor, preferentially generated over its optical isomer, (8R,9S)-EET, suggesting that it is biologically relevant and implying specific structural requirements for EET receptor activation. The principal mechanism of action of 8,9-EET is preglomerular vasoconstriction. The vasoconstrictor effect of 8,9-EET is CO dependent.