Background:Reasons for the differences in activity between the peroxisomal and mitochondrial DCRs are unknown. Results: Structure-function studies on peroxisomal DCR (pDCR) and comparison with its mitochondrial counterpart reveal differences in catalytic residues and hinge movements. Conclusion: Catalysis by pDCR is unique and involves aspartate. Significance: Our studies explain why human peroxisomal DCR, but not mitochondrial DCR, can process very long chain fatty acids.