The biotransformation in human blood in vitro of three dynorphin A (Dyn A) peptides was studied by matrix assisted laser desorption mass spectrometry to determine whether the natural peptide, Dyn A(1-17), is biotransformed differently from Dyn A (1-13), the natural sequence shortened form used in numerous neurobiological and pharmacological studies. In addition to studies of Dyn A(1-17), a natural product from prodynorphin and Dyn A(1-13), a natural sequence truncation of Dyn A(1-17), Dyn A(1-10)amide, a synthetic analogue of Dyn A(1-17) presumed to be protected from rapid biotransformation was also studied Synthetic Dyn A peptides were incubated in freshly drawn blood for various periods of time prior to mass spectrometric analysis. Several peptide products were identified from each precursor; the time profiles of appearance and disappearance of the major products were followed. Substantial differences in products and especially in the rate of biotransformation were observed between the processing of Dyn A(1-17) and the two shorter Dyn A peptides, Dyn A(1-13) and Dyn A(1-10)amide. Significant amounts of the natural Dyn A(1-17) survived 4 h of incubation (half-life 3 h). Dyn A (2-17), a major processed product of Dyn A(1-17) in blood, continued to accumulate during the 4-h incubation period. By contrast, both Dyn A(1-13) and Dyn A(1-10) amide were biotransformed very rapidly with half-lives of < 1 min and 10 min, respectively. Most of the products from these two peptide precursors were also further processed rapidly, with the exception of Dyn A(4-12) and Dyn A(4-10)amide, which were detected for over 2 h. Dyn A(1-6) was found as a minor biotransformation product from all three precursor peptides. These findings suggest that an important function of the four C-terminal amino acid residues of the natural form, Dyn A(1-17) [compared to Dyn A(1-13)], is to stabilize or protect the peptide from biotransformation by enzymes, by preserving a natural hairpin structure possibly near the carboxyl-terminus.