In order to clarify the physiological significance of stereospecificities of peroxisomal multifunctional enzyme (MFE) type 1 (MFE1) and MFE2, we developed a chiral separation analysis for 3-hydroxyacyl-CoA using high performance liquid chromatography (HPLC) equipped with a chiral separation column. To demonstrate the utility of this technique, we cloned the hydratase domain from wild-type human MFE2 hydratase (MFE2Hwt) and expressed it as a GFP-tagged protein (GFP-MFE2Hwt) in Escherichia coli (E. coli). GFP-MFE2H was purified by diethylaminoethyl (DEAE) Sephacel from an E. coli sonication solution. As anticipated, we observed the formation of 3R-hydroxyhexadecanoyl-CoA (3R-OH-16-CoA) on the HPLC chromatogram after incubating trans-2-enoyl-CoA (16eno-CoA) with GFP-MFE2Hwt. GFP-MFE2Hwt was readily purifiable and could be assayed because of its traceability. We used site-directed mutagenesis to construct GFP-MFE2H variants corresponding to 17 reported MFE2H missense mutations and measured their hydratase activities using our HPLC method. Hydratase activity was completely lost or markedly decreased in the same variants corresponding to MFE2H mutations in patients with D-bifunctional protein (DBP) deficiency type II. On the other hand, the nonpathological variants did not markedly affect hydratase activity.