The in vivo administration of certain monoclonal antibodies (mAbs) against the adhesion receptor, CD44, into normal mice induces both a modulation of CD44 from the surface of peripheral lymphocytes, and a concomitant increase in the amount of soluble CD44 in the serum. CD44-negative lymphocytes isolated from anti-CD44-treated mice exhibit normal homing patterns upon adoptive transfer, and are capable of reexpressing CD44 upon activation. The treatment of haptensensitized mice with anti-CD44 mAb inhibits their ability to mount a cutaneous delayed-type hypersensitivity (DTH) response within the first 24 h after hapten challenge. This inhibition reflects a block in both the edema and leukocyte infiltration of the cutaneous site of DTH, whereas the extravasation and accumulation of leukocytes in the draining lymph nodes progress normally. After 72 h, the leukocytes that extravasate into the site of antigen challenge express CD44. These results indicate that CD44 is not necessary for normal leukocyte circulation but is required for leukocyte extravasation into an inflammatory site involving nonlymphoid tissue.