Ubiquitin-peptide fusion protein system enables preparation of stable isotope labeled peptides through the expression of the protein in E. coli cells in labeled media (Kohno et al. (1998) J Biomol NMR 12:109–121). Advantages of the system over others include: very specific cleavage of the bond between ubiquitin and target peptide moieties by yeast ubiquitin hydrolase and low cost for the protease which can also be expressed in E. coli cells. The former point is particularly important since other frequently used proteases, such as factor Xa and thrombin, often show non-specific cleavages at sites unexpected from their nominal specificities. We improved the yield of the peptide by adapting the codon usage of ubiquitin gene for the expression in E. coli cells, by using RNase E-deficient host strains, and by modifying purification procedure. The yield of mastoparan-X was increased threefold by these modifications. We also succeeded in the preparation of labeled magainin 2, an antimicrobial peptide that could not be expressed at all by the previous method, by choosing host strains and culture media. The HSQC signals of the 15N-labeled magainin 2 in an aqueous solution were completely resolved in spite of the severe overlap of the 1D proton signals, confirming that the stable isotope labeling is quite useful for analysis of peptides.