During inflammation, activated monocytes (Mo) migrate into tissues where they interact with extracellular matrix components such as hyaluronate (HA), produced in high amounts at inflammatory sites. We determined whether Mo that had invaded sites of cutaneous inflammation bind HA and express the putative HA receptors CD44 isoforms, ICAM-1, or receptor for hyaluronate-mediated motility (RHAMM). In cutaneous inflammation, activated infiltrating Mo displayed high HA avidity and expressed epitopes encoded by CD44s, CD44 variant exons v3, v4, v5, v6, v7, and v9, and ICAM-1, but not RHAMM. We further investigated how activation affects the avidity of Mo for HA and which receptors were responsible for such binding. Mo freshly purified from human peripheral blood bound little HA and expressed CD44s but no epitopes encoded by CD44v exons, ICAM-1, or RHAMM. During short-term tissue culture, Mo upregulated their HA avidity and expression of ICAM-1, CD44s, and epitopes encoded by CD44v, all of which were further augmented by IFN-gamma or lipopolysaccharide, whereas RHAMM was not detectable. Thus in vitro activated Mo resembled Mo that had migrated to inflammatory sites in vivo. Lipolysaccharide or IFN-gamma-induced HA binding was inhibited by more than 90% with monoclonal antibodies directed against N-terminal HA binding domains of CD44s, but not by monoclonal antibodies against CD44v epitopes or ICAM-1. In conclusion, we show that upon in vitro or in vivo activation, Mo enhance their capacity to bind HA. This is critically dependent upon the expression ofCD44s epitopes. Regulated CD44-HA interactions may be important for the ability of Mo to migrate into and within sites of inflammation and for Mo effector functions.