CD26 is an antigen with key role in T-cell biology and is expressed on selected subsets of aggressive T-cell malignancies. To elucidate the role of CD26 in tumor behavior, we examine the effect of CD26 depletion by small interfering RNA transfection of T-anaplastic large cell lymphoma Karpas 299. We show that the resultant CD26-depleted clones lose the ability to adhere to fibronectin and collagen I. Because anti-integrin beta1 blocking antibodies also prevent binding of Karpas 299 to fibronectin and collagen I, we then evaluate the CD26-integrin beta1 association. CD26 depletion does not decrease integrin beta1 expression but leads to dephosphorylation of both integrin beta1 and p38 mitogen-activated protein kinase (MAPK). Moreover, our data showing that the p38MAPK inhibitor SB203580 dephosphorylates integrin beta1 and that binding of the anti-CD26 antibody 202.36 dephosphorylates both p38MAPK and integrin beta1 on Karpas 299, leading to loss of cell adhesion to the extracellular matrix, indicate that CD26 mediates cell adhesion through p38MAPK-dependent phosphorylation of integrin beta1. Finally, in vivo experiments show that depletion of CD26 is associated with loss of tumorigenicity and greater survival. Our findings hence suggest that CD26 plays an important role in tumor development and may be a novel therapeutic target for selected neoplasms.