Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging， drug discovery， diagnostics， and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20， an antimicrobial peptide from the C-terminus of human thrombin， (C)ApoB， an antimicrobial peptide from an inner region of human Apolipoprotein B， and (C)p53pAnt， an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH， temperature， and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY， GTGDCHVA， GSGTDCGSR， SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures)， high purity (>96%)， and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore， (C)GKY20 was modified with PyMPO-maleimide， a commercially available fluorophore bearing a thiol reactive group， and with 6-hydroxy-2-cyanobenzothiazole， a reagent specific for N-terminal cysteines， with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.