In the present study, we investigated the involvement of CD44 in leukocyte trafficking in vivo at the blood-retinal barrier using experimental autoimmune uveoretinitis (EAU) as a model system. Leukocyte trafficking was evaluated using adoptive transfer of calcein-AM (C-AM)-labeled spleen cells harvested from syngeneic mice at prepeak severity of EAU to mice at a similar stage of disease. CD44 and its ligand hyaluronan were up-regulated in the eye during EAU. CD44-positive leukocytes were found sticking in the retinal venules and postcapillary venules but not in the retinal arterioles nor in mesenteric vessels. Preincubation of in vitro C-AM-labeled leukocytes with anti-CD44 monoclonal antibodies (mAb; IM7) or high molecular weight hyaluronic acid (HA) before transfer significantly suppressed leukocyte rolling but not sticking in retinal venules and also reduced cell infiltration in the retinal parenchyma. Administration of the HA-specific enzyme hyaluronidase to mice before cell transfer also reduced leukocyte infiltration, suggesting that CD44-HA interactions are involved in leukocyte recruitment in EAU. This was further supported by the observation that disease severity was reduced by administration of anti-CD44 mAb (IM7) at the early leukocyte-infiltration stage. Further studies also indicated that CD44 activation was associated with increased levels of apoptosis, and this may also be in part responsible for the reduction in disease severity. These findings demonstrate that CD44 is directly involved in leukocyte-endothelial interaction in vivo and influence the trafficking of primed leukocytes to the retina and their overall survival.