Fibronectin (FN) forms meshworks in extracellular spaces, and it plays an important role in cellular trafficking. Lymphoid cells are activated by binding to FN of the VLA-4 and VLA-5 receptors. CD44 also acts as a receptor of FN, but the mechanism and physiologic regulation of their binding are poorly understood. We have developed an anti-CD44 monoclonal antibody (mAb) (TL-1) in which lymphoid cells are activated and form homotypic cell aggregation. In this study, we found that the adhesion of CEM, HSB2, and LAD lymphoid cells to FN was augmented by TL-1 treatment and was apparently blocked by another anti-CD44 mAb (Hermes-3), but TL-1 Fab' fragments treatment did not induce FN-binding. A similar phenomenon is reported in the binding of the CD44 molecule to HA. This augmentation was not inhibited by the CS1 and RGD peptides of FN or by anti-VLA-4 and -VLA-5 mAbs; it was energy-dependent and associated with cytoplasmic actin filaments. Tl-1 treatment did not alter the cell surface expression of CD44 molecules. These findings above suggested that activated and/or altered cell surface distribution of CD44 molecules via a conformational change augmented the avidity of its binding to FN, which may be similar to lymphocyte-hyaluronate and lymphocyte-endothelial cell binding. As the Hermes-3 binding site is also involved in the interaction between lymphocytes and endothelial cells, activation of lymphocytes via CD44 molecules may facilitate the binding of lymphocytes to endothelial cells, extravasation, and migration to inflammatory sites rich in FN.