CD44 is a polymorphic glycoprotein expressed on the surface of many tissues and cell lines which has been implicated in a number of cellular functions including lymphocyte homing to mucosal lymphoid tissue (Peyers patches), leukocyte activation, lymphopoiesis, and tumor metastasis. The predominant isoform found on human leukocytes, CD44H, is a receptor for hyaluronic acid. Because of the prominent role CD44 plays in diverse biological processes, we set out to identify the hyaluronic acid binding site(s) in the extracellular domain of CD44H. Using truncation and site-directed mutagenesis we identified two regions containing clusters of conserved basic residues which are important in hyaluronic acid binding. One of these regions is situated near the NH2 terminus and is homologous to other hyaluronic acid binding proteins including cartilage link protein. The other more membrane proximal region lies outside the link protein homologous domain. Mutagenesis of basic residues within these regions established their role as determinants in hyaluronic acid binding. Mutation of Arg 41, a position where a basic residue is conserved in all hyaluronic acid binding proteins, completely abolished binding suggesting that this residue plays a critical role in hyaluronic acid binding.