Epoxyeicosatrienoic acids (EETs) are potent regulators of vascular homeostasis and are bound by cytosolic fatty acid-binding proteins (FABPs) with K(d) values of approximately 0.4 microM. To determine whether FABP binding modulates EET metabolism, we examined the effect of FABPs on the soluble epoxide hydrolase (sEH)-mediated conversion of EETs to dihydroxyeicosatrienoic acids (DHETs). Kinetic analysis of sEH conversion of racemic [(3)H]11,12-EET yielded K(m) = 0.45 +/- 0.08 microM and V(max) = 9.2 +/- 1.4 micromol min(-1) mg(-)(1). Rat heart FABP (H-FABP) and rat liver FABP were potent inhibitors of 11,12-EET and 14,15-EET conversion to DHET. The resultant inhibition curves were best described by a substrate depletion model, with K(d) = 0.17 +/- 0.01 microM for H-FABP binding to 11,12-EET, suggesting that FABP acts by reducing EET availability to sEH. The EET depletion by FABP was antagonized by the co-addition of arachidonic acid, oleic acid, linoleic acid, or 20-hydroxyeicosatetraenoic acid, presumably due to competitive displacement of FABP-bound EET. Collectively, these findings imply that FABP might potentiate the actions of EETs by limiting their conversion to DHET. However, the effectiveness of this process may depend on metabolic conditions that regulate the levels of competing FABP ligands.