Two rapid assays for the soluble epoxide hydrolase (sEH) are described. First, a sensitive radiometric assay based on thin-layer chromatography of [(14)C]-cis-9,10-epoxystearic acid and its corresponding diol ((14)C]-9,10-dihydroxystearic acid) is described. The cis fatty acid oxide exhibits higher specific activity of hydration with sEH from mouse, rat, human, and potato compared to trans-stilbene oxide (TSO). The K(m) and V(max) obtained for [(14)C]-cis-9,10-epoxystearic acid with mouse sEH are 11.0 microM and 3460 nmol/min/mg protein, respectively. [(14)C]-cis-9,10- Epoxystearic acid might more closely mimic the structures of natural substrates for sEH. Second, [2-(3)H]-trans-1,3-diphenyl-propene oxide ([(3)H]-tDPPO) and [2-(3)H]-cis-1,3-diphenylpropene oxide ([(3)H]-cDPPO) were synthesized and rapid radiometric assays for epoxide hydrolases (EHs) were developed by differential partitioning of the epoxide into iso-octane and its corresponding diol into aqueous phase containing methanol. It was shown that sEHs from mouse, rat, human, and potato rapidly hydrolyze [(3)H]-tDPPO and in comparison to TSO have 20-,49-,28-, and 7-fold higher rates, respectively. Mouse sEH hydrates [(3)H]-tDPPO at 26,200 nmol/min/mg protein, and a K(m)p4 of 2.80 microM is observed.