Abstract The interferon (IFN)–inducible chemokines, specifically, IFN-γ–inducible protein-10 (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α-chemoattractant (I-TAC), share a unique CXC chemokine receptor (CXCR3). Recently, the highly specific membrane-bound protease and lymphocyte surface marker CD26/dipeptidyl peptidase IV (DPP IV) was found to be responsible for posttranslational processing of chemokines. Removal of NH2-terminal dipeptides by CD26/DPP IV alters chemokine receptor binding and signaling, and hence inflammatory and anti–human immunodeficiency virus (HIV) activities. CD26/DPP IV and CXCR3 are both markers for Th1 lymphocytes and, moreover, CD26/DPP IV is present in a soluble, active form in human plasma. This study reports that at physiologic enzyme concentrations CD26/DPP IV cleaved 50% of I-TAC within 2 minutes, whereas for IP-10 and Mig the kinetics were 3- and 10-fold slower, respectively. Processing of IP-10 and I-TAC by CD26/DPP IV resulted in reduced CXCR3-binding properties, loss of calcium-signaling capacity through CXCR3, and more than 10-fold reduced chemotactic potency. Moreover, IP-10 and I-TAC cleaved by CD26/DPP IV acted as chemotaxis antagonists and CD26/DPP IV–truncated IP-10 and Mig retained their ability to inhibit the angiogenic activity of interleukin-8 in the rabbit cornea micropocket model. These data demonstrate a negative feedback regulation by CD26/DPP IV in CXCR3-mediated chemotaxis without affecting the angiostatic potential of the CXCR3 ligands IP-10 and Mig.