Objective: Peroxisomes are single membrane eukaryotic organelles that perform variable functions. They share a common biogenetic mechanism but have different functions depending on the tissue, the developmental stage or environmental conditions. Peroxisomes are found in all eukaryotic cells and stem cells.Their proteins are encoded by the nuclear genome and imported from the cytoplasm. Peroxisomal matrix proteins are synthesized in the cytoplasm and targeted to peroxisomes by virtue of a peroxisomal targeting signal (PTS). One of the peroxisomal matrix proteins -Peroxisomal Protein (PeP)- has shown different pattern of expression in mouse embryo in various tissues, but the reason is unclear. PeP cDNA was cloned and then subcloned in pGEX6p2 prokaryotic expression vector in order to label this gene with GST to purify PeP protein for further biochemical analysis and identifying related proteins. PeP was inserted downstream of FLAG gene in pUCD3-FLAG eukaryotic expression vector to express tagged-PeP protein for transient transfection analysis and identifying localization of PEP protein. Materials and Methods: PEP-cDNA was amplified in different PCR reactions using pEGFP-PEP vector and 2 sets of primers introducing specific restriction sites at the ends of PEP. PCR products with BamHI/SalI restriction sites were treated by restriction enzymes and inserted into the pGEX6p2, downstream of GST tag. PEP-cDNA containing BamHI/ApaI restriction sites and FLAG gene -which amplified using pUcD3-FLAGPEX3 vector- were used as templates in secondary PCR for amplifying FLAG-PEP recombinant DNA. FLAGPEP fragment was treated by enzymatic digestion and inserted into the pUcD3 eukaryotic expression vector. pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells respectively. The positive colonies were selected for plasmid preparation and additional analysis. . Finally, to confirm the intracellular localization of FLAG-PEP, Chinese hamster ovary (CHO) and P19 cells were transfected with the constructed plasmid. Results: Our results confirmed amplification of the expected products. PEP-cDNA in both PCR reactions encompasses 630bp which encodes 209 amino acid residues. FLAG fragment containing designed sites was 77bp and FLAG-PEP fragment was 700bp. Sequencing of constructed vectors confirms that PEP-cDNA was tagged appropriately and inserted free of mutation and in frame with GST and FLAG. Because of the presence of SKI in the C-terminal of the related protein, transfection data showed peroxisomal localization of FLAGPEP as was similar to the catalase. Taken together these data confirmed that PEP is a peroxisomal protein. Conclusion: We have sub-cloned PEP-cDNA in prokaryotic and eukaryotic expression vectors to tag it with GST and FLAG tandems. Previous studies have indicated that PEP protein is a peroxisomal protein. Transient transfection in CHO and P19 cells with constructed vector confirmed peroxisomal localization of related protein.