Recombinant expression of α-defensins can be obtained at efficient levels in Escherichia coli. Amplified α-defensin or pro-α-defensin coding cDNA sequences are cloned directionally between EcoRI and SalI sites of the pET-28a expression vector and expressed in E. coli BL21 RIS cells. Cells growing exponentially in nutrient-rich liquid medium are induced to express the recombinant protein by addition of 50 mM isopropyl β-d-1-thiogalactopyranoside for 3–6 h. After bacterial cells collected by centrifugation are lysed in 6 M guanidine-HCl under non-reducing conditions, the expressed defensin fused to its 6xHis-34 amino acid N-terminal fusion partner is purified by affinity chromatography on nickel-NTA columns. A Met codon introduced at the N terminus of expressed Met-free peptides provides a unique CNBr cleavage site, enabling release of the α-defensin free of ancillary residues by sequential C18 RP-HPLC. Molecular masses of C18 RP-HPLC purified peptides are confirmed by MALDI-TOF mass spectrometry, and peptide homogeneity is assessed using analytical RP-HPLC and acid-urea polyacrylamide gel electrophoresis. α-Defensins prepared in this manner are biochemically equivalent to the natural molecules.