Abstract A soluble enzyme preparation from Pseudomonas sp. NRRL B-3266 catalyzes the interconversion of cis-Δ9-octadecenoic acid (oleic acid), trans-Δ10-octadecenoic acid, and 10-d-hydroxyoctadecanoic acid. Oleic acid and 10-d-hydroxy-octadecanoic acid are directly interconvertible by hydration and dehydration. The cis and trans unsaturated fatty acid isomers are interconvertible by direct isomerization of the double bond. trans-Δ10-Octadecenoic and 10-d-hydroxyoctadecanoic acid do not undergo direct interconversion without the intermediate formation of (enzyme-bound) oleic acid. Both hydration and isomerization reactions are very specific for a cis-Δ9 double bond, but the isomerization reaction is much more sensitive to changes in the hydrocarbon tail of the substrate distal to the double bond. The isomerization proceeds stereospecifically, with the 11-l (Pro-R) hydrogen of oleic acid being removed and a hydrogen from the medium being added at carbon 9 in the l (Pro-R) configuration. In the reverse reaction the 9-l (Pro-R) hydrogen is removed and a hydrogen from the medium is added at carbon 11. Primary kinetic isotope effects are seen for the removal of the 9-l and 11-l hydrogens. There is a marked discrimination against tritium in the addition of the 11-l hydrogen from the medium. The hydration of oleic acid and the isomerization of trans-Δ10-octadecenoic acid to oleic acid proceed more slowly in highly enriched 2H2O than in H2O, but the isomerization of oleic acid to trans-Δ10-octadecenoic acid is slightly faster in 2H2O.