The protease-catalyzed conversion of peptides and proteins produced by recombinant DNA technology is a promising method for large-scale production of peptides including those with non-proteinogenic structural elements. As a model system we have investigated the proteinase-catalyzed modification of a chemically synthesized substance P-precursor. In the precursor peptide the residues of substance P(1-8) were flanked by tripeptide linkers on both sides. In the first step the C-terminal tripeptide amide was replaced by the authentic C-terminal tripeptide amide of substance P via alpha-chymotrypsin-catalyzed transpeptidation. The enzyme simultaneously attacks two peptide bonds in the precursor molecule leading to the formation of several side-products. The desired peptide was obtained with 25% yield. In the second step the other tripeptide linker was selectively and almost quantitatively removed from the N-terminus of the precursor via trypsin-catalyzed hydrolysis. This study demonstrates that substance P can be obtained from an engineered protein by proteinase-catalyzed processing.