Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease hydrolyzing the release of dipeptides from the N-terminus of oligopeptides and small polypeptides. The proline specific enzyme can either inactivate or activate by its catalytic action, a variety of peptides. This work deals with structural and functional relation studies of DP IV regarding its hydrolytic action on substrates (physiological substrates and dipeptide derivatives) and inhibitor binding after previously establishing and optimizing the expression and purification of human recombinant DP IV. Human DP IV was expressed in Pichia pastoris in a fermentation set up, purified to homogeneity and characterized. In the future, the optimized expression and purification procedure will allow kinetic characterization studies of inhibitors and substrates to be performed with human recombinant DP IV. In order to elucidate the function and the contribution of single amino acids, such as R125, E205, E206, Y547, W629, Y631 and N710, which surround the catalytic triad of DP IV, their specific role was investigated by carrying out mutagenesis studies. It was shown, that the kinetics found with synthetic dipeptide derivatives was not the same as that observed with physiological substrates. The amino acid N710 and R125 play an essential role in the binding and hydrolysis of long natural substrates with alanine (GIP) or serine (PACAP38) in the P1-position while substrates with proline in this position are not dependent on the amino acids R125 and N710. Based on the findings that a certain chain length is necessary to stabilize peptide binding, possible inhibition of these "secondary" interactions was investigated. A new class of inhibitor directed against "secondary" interactions between enzyme and substrate was generated which allows for the first time to distinguish between substrate families avoiding putative severe side effects in therapy, based on the inhibition of DP IV catalysis. During the characterization studies, it was demonstrated, that the pancreatic polypeptide (36 amino acids of length, proline in the P1-position), a member of the pancreatic polypeptide family, is a substrate of DP IV. A dataset with a resolution of 2.08 Å was obtained from protein crystal analysis by X-Ray Diffraction experiments with human recombinant DP IV. It could be demonstrated, that Humanin (HN) (consists of 24 amino acids), a recently described intracellular located peptide with a protective effect against Bax induced cell death, is processed by PEP. In addition, unexpected post-cysteine(8) specific hydrolysation of HN was observed. The previously undiscovered specificity for a post-cysteine cleavage was also demonstrated with DP IV, by processing the GIP variant A2C-GIP after the cysteine residue.