Dipeptidyl peptidase IV-β (DPP IV-β) is a novel protein which shows a peptidase activity similar to the T-cell-activation antigen CD26. To further characterize this DPP IV-β and confirm its cell surface expression, we have developed a purification strategy using the CD26 Ϫ cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel-filtration, ion-exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV-β estimated by gel-filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75Ϫ80-kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV-β and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different K m values for Gly-Pro-NH-Np (NH-Np, para-nitroanilide) hydrolysis (0.28 Ϯ 0.05 mM and 0.12Ϯ0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-β. These differences permitted us to study DPP IV-β expression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin-stimulated or protein-A-stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV-β and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.