SummaryHuman transforming growth factor-b3 (TGFb3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFb3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFb3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFb3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFb3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFb3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFb3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFb3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFb3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFb3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFb3.