Peptides of the glucagon/vasoactive-intestinal-peptide (VIP) peptide family share a considerable sequence similarity at their N-terminus. They either start with Tyr-Ala, His-Ala or His-Ser which might be in part potential targets for dipeptidyl-peptidase IV, a highly specialized aminopeptidase removing dipeptides only from peptides with N-terminal penultimate proline or alanine. Growthhormone-releasing factor(1–29)amide and gastric inhibitory peptide/glucose-dependent insulinotropic peptide (GIP) with terminal Tyr-Ala as well as glucagon-like peptide-1(7–36)amide/insulinotropin [GLP-1(7–36)amide] and peptide histidine methionine (PHM) with terminal His-Ala were hydrolysed to their des-Xaa-Ala derivatives by dipeptidyl-peptidase IV purified from human placenta. VIP with terminal His-Ser was not significantly degraded by the peptidase. The kinetics of the hydrolysis of GIP, GLP-1(7–36)amide and PHM were analyzed in detail. For these peptides Km values of 4–34 μM and Vmax values of 0.6–3.8 μmol · min−1· mg protein−1 were determined for the purified peptidase which should allow their enzymic degradation also at physiological, nanomolar concentrations. When human serum was incubated with GIP or GLP-1(7–36)amide the same fragments as with the purified dipeptidyl-peptidase IV, namely the des-Xaa-Ala peptides and Tyr-Ala in the case of GIP or His-Ala in the case of GLP-1(7–36)amide, were identified as the main degradation products of these peptide hormones. Incorporation of inhibitors specific for dipeptidylpeptidase IV, 1 mM Lys-pyrrolidide or 0.1 mM diprotin A (Ile-Pro-Ilc), completely abolished the production of these fragments by serum. It is concluded that dipeptidyl-peptidase IV initiates the metabolism of GIP and GLP-1(7–36)amide in human serum. Since an intact N-terminus is obligate for the biological activity of the members of the glucagon/VIP peptide family [e. g. GIP(3–42) is known to be inactive to release insulin in the presence of glucose as does intact GIP], dipeptidyl-peptidase-IV action inactivates these peptide hormones. The relevance of this finding for their inactivation and their determination by immunoassays is discussed.